Prevention and treatment of amyloidogenic disease

ABSTRACT

The invention provides compositions and methods for treatment of amyloidogenic diseases. Such methods entail administering an agent that induces a beneficial immune response against an amyloid deposit in the patient. The methods are particularly useful for prophylactic therapeutic treatment of Alzheimer&#39;s disease. In such methods, a suitable agent is Aβ peptide or an antibody thereto.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/201,430, filed Nov. 30, 1998, which claims the benefit under 35U.S.C. 119(e) of U.S. application Ser. No. 60/080,970, filed Apr. 7,1998 and U.S. application Ser. No. 60/067,740, filed Dec. 2, 1997, allof which are incorporated by reference in their entirety for allpurposes.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a progressive disease resulting in seniledementia. See generally Selkoe, TINS 16, 403-409 (1993); Hardy et al.,WO 92/13069; Selkoe, J. Neuropathol. Exp. Neurol. 53, 438-447 (1994);Duff et al., Nature 373, 476-477 (1995); Games et al., Nature 373, 523(1995). Broadly speaking the disease falls into two categories: lateonset, which occurs in old age (65+ years) and early onset, whichdevelops well before the senile period, i.e, between 35 and 60 years. Inboth types of disease, the pathology is the same but the βnormalitiestend to be more severe and widespread in cases beginning at an earlierage. The disease is characterized by two types of lesions in the brain,senile plaques and neurofibrillary tangles. Senile plaques are areas ofdisorganized neuropil up to 150 μn across with extracellular amyloiddeposits at the center visible by microscopic analysis of sections ofbrain tissue. Neurofibrillary tangles are intracellular deposits of tauprotein consisting of two filaments twisted about each other in pairs.

The principal constituent of the plaques is a peptide termed Aβ orβ-amyloid peptide. Aβ peptide is an internal fragment of 39-43 aminoacids of a precursor protein termed amyloid precursor protein (APP).Several mutations within the APP protein have been correlated with thepresence of Alzheimer's disease. See, e.g., Goate et al., Nature 349,704) (1991) (valine⁷¹⁷ to isoleucine); Chartier Harlan et al. Nature353, 844 (1991)) (valine⁷¹⁷ to glycine); Murrell et al., Science 254, 97(1991) (valine⁷¹⁷ to phenylalanine); Mullan et al., Nature Genet. 1, 345(1992) (a double mutation changing lysine⁵⁹⁵-methionine⁵⁹⁶ toasparagine⁵⁹⁵-leucine⁵⁹⁶). Such mutations are thought to causeAlzheimer's disease by increased or altered processing of APP to Aβ,particularly processing of APP to increased amounts of the long form ofAβ (i.e., Aβ1-42 and Aβ1-43). Mutations in other genes, such as thepresenilin genes, PS1 and PS2, are thought indirectly to affectprocessing of APP to generate increased amounts of long form Aβ (seeHardy, TINS 20, 154 (1997)). These observations indicate that Aβ, andparticularly its long form, is a causative element in Alzheimer'sdisease.

McMichael, EP 526,511, proposes administration of homeopathic dosages(less than or equal to 10⁻² mg/day) of Aβ to patients withpreestablished AD. In a typical human with about 5 liters of plasma,even the upper limit of this dosage would be expected to generate aconcentration of no more than 2 pg/ml. The normal concentration of Aβ inhuman plasma is typically in the range of 50-200 pg/ml (Seubert et al.,Nature 359, 325-327 (1992)). Because EP 526,511's proposed dosage wouldbarely alter the level of endogenous circulating Aβ and because EP526,511 does not recommend use of an adjuvant, it seems implausible thatany therapeutic benefit would result.

By contrast, the present invention is directed inter alia to treatmentof Alzheimer's and other amyloidogenic diseases by administration of Aβor other immunogen to a patient under conditions that generate abeneficial immune response in the patient. The invention thus fulfills alongstanding need for therapeutic regimes for preventing or amelioratingthe neuropathology of Alzheimer's disease.

SUMMARY OF THE CLAIMED INVENTION

In one aspect, the invention provides methods of preventing or treatinga disease characterized by amyloid deposition in a patient. Such methodsentail inducing an immune response against a peptide component of anamyloid deposit in the patient. Such induction can be active byadministration of an immunogen or passive by administration of anantibody or an active fragment or derivative of the antibody. In somepatients, the amyloid deposit is aggregated Aβ peptide and the diseaseis Alzheimer's disease. In some methods, the patient is asymptomatic. Insome methods, the patient is under 50 years of age. In some methods, thepatient has inherited risk factors indicating susceptibility toAlzheimer's disease. Such risk factors include variant alleles inpresenilin gene PS1 or PS2 and variant forms of APP. In other methods,the patient has no known risk factors for Alzheimer's disease.

For treatment of patients suffering from Alzheimer's disease, onetreatment regime entails administering a dose of Aβ peptide to thepatient to induce the immune response. In some methods, the Aβ peptideis administered with an adjuvant that enhances the immune response tothe Aβ peptide. In some methods, the adjuvant is alum. In some methods,the adjuvant is MPL. The dose of Aβ peptide administered to the patientis typically at least 1 or 10 μg, if administered with adjuvant, and atleast 50 μg if administered without adjuvant. In some methods, the doseis at least 100 μg.

In some methods, the Aβ peptide is Aβ1-42. In some methods, the Aβpeptide is administered in aggregated form.

In other methods, the Aβ peptide is administered in dissociated form. Insome methods, the therapeutic agent is an effective dose of a nucleicacid encoding Aβ or an active fragment or derivative thereof. Thenucleic acid encoding Aβ or fragment thereof is expressed in the patientto produce Aβ or the active fragment thereof, which induces the immuneresponse. In some such methods, the nucleic acid is administered throughthe skin, optionally via a patch. In some methods, a therapeutic agentis identified by screening a library of compounds to identify a compoundreactive with antibodies to Aβ, and administering the compound to thepatient to induce the immune response.

In some methods, the immune response is directed to aggregated Aβpeptide without being directed to dissociated Aβ peptide. For example,the immune response can comprise antibodies that bind to aggregated Aβpeptide without binding to dissociated Aβ peptide. In some methods, theimmune response comprises T-cells that bind to Aβ complexed with MCH1 orMHCII on CD8 or CD4 cells. In other methods, the immune response isinduced by administering an antibody to Aβ to the patient. In somemethods, the immune response is induced by removing T-cells from thepatient, contacting the T-cells with Aβ peptide under conditions inwhich the T-cells are primed, and replacing the T-cells in the patient.

The therapeutic agent is typically administered orally, intranasally,intradermally, subcutaneously, intramuscularly, topically orintravenously. In some methods, the patient is monitored followedadministration to assess the immune response. If the monitoringindicates a reduction of the immune response over time, the patient canbe given one or more further doses of the agent.

In another aspect, the invention provides pharmaceutical compositionscomprising Aβ and an excipient suitable for oral and other routes ofadministration. The invention also provides pharmaceutical compositionscomprising an agent effective to induce an immunogenic response againstAβ in a patient, and a pharmaceutically acceptable adjuvant. In somesuch compositions, the agent is Aβ or an active fragment thereof. Insome compositions, the adjuvant comprises alum. In some compositions,the adjuvant comprises an oil-in-water emulsion. In some compositions,the Aβ or active fragment is a component of a polylactide polyglycolidecopolymer (PLPG) or other particle. The invention further providescompositions comprising Aβ or an active fragment linked to a conjugatemolecule that promotes delivery of Aβ to the bloodstream of a patientand/or promotes an immune response against Aβ. For example, theconjugate can serve to promote an immune response against Aβ. In somecompositions, the conjugate is cholera toxin. In some compositions, theconjugate is an immunoglobulin. In some compositions, the conjugate isattenuated diphtheria toxin CRM 197 (Gupta, Vaccine 15, 1341-3 (1997).

The invention also provides pharmaceutical compositions comprising anagent effect to induce an immunogenic response against Aβ in a patientwith the proviso that the composition is free of Complete Freund'sadjuvant. The invention also provides compositions comprising a viralvector encoding Aβ or a an active fragment thereof effective to inducean immune response against Aβ. Suitable viral vectors include herpes,adenovirus, adenoassociated virus, a retrovirus, sindbis, semilikiforest virus, vaccinia or avian pox.

The invention further provides methods of preventing or treatingAlzheimer's disease. In such methods, an effective dose of Aβ peptide isadministered to a patient. The invention further provides for the use ofAβ, or an antibody thereto, in the manufacture of a medicament forprevention or treatment of Alzheimer's disease.

In another aspect, the invention provides methods of assessing efficacyof an Alzheimer's treatment method in a patient. In these methods, abaseline amount of antibody specific for Aβ peptide is determined in atissue sample from the patient before treatment with an agent. An amountof antibody specific for Aβ peptide in the tissue sample from thepatient after treatment with the agent is compared to the baselineamount of Aβ peptide-specific antibody. An amount of Aβ peptide-specificantibody measured after the treatment that is significantly greater thanthe baseline amount of Aβ peptide-specific antibody indicates a positivetreatment outcome.

In others methods of assessing efficacy of an Alzheimer's treatmentmethod in a patient, a baseline amount of antibody specific for Aβpeptide in a tissue sample from a patient before treatment with an agentis determined. An amount of antibody specific for Aβ peptide in thetissue sample from the subject after treatment with the agent iscompared to the baseline amount of Aβ peptide-specific antibody. Areduction or lack of significant difference between the amount of Aβpeptide-specific antibody measured after the treatment compared to thebaseline amount of Aβ peptide-specific antibody indicates a negativetreatment outcome.

In other methods of assessing efficacy of an Alzheimer's diseasetreatment method in a patient a control amount of antibody specific forAβ peptide is determined in tissue samples from a control population.,An amount of antibody specific for Aβ peptide in a tissue sample fromthe patient after administering an agent is compared to the controlamount of Aβ peptide-specific antibody. An amount of Aβ peptide-specificantibody measured after the treatment that is significantly greater thanthe control amount of Aβ peptide-specific antibody indicates a positivetreatment outcome.

In other methods of assessing efficacy of an Alzheimer's treatmentmethod in a patient, a control amount of antibody specific for Aβpeptide in tissues samples from a control population is determined. Anamount of antibody specific for Aβ peptide in a tissue sample from thepatient after administering an agent is compared to the control amountof Aβ peptide-specific antibody. A lack of significant differencebetween the amount of Aβ peptide-specific antibody measured afterbeginning said treatment compared to the control amount of Aβpeptide-specific antibody indicates a negative treatment outcome.

Other methods of monitoring Alzheimer's disease or susceptibilitythereto in a patient, comprise detecting an immune response against Aβpeptide in a sample from the patient. In some such methods, the patientis being administered an agent effective to treat or prevent Alzheimer'sdisease, and the level of the response determines the future treatmentregime of the patient.

In other methods of assessing efficacy of an Alzheimer's treatmentmethod in a patient a value for an amount of antibody specific for Aβpeptide in tissue sample from a patient who has been treated with anagent is determined. The value is compared with a control valuedetermined from a population of patient experiencing amelioriation of,or freedom from, symptoms of Alzheimer's disease due to treatment withthe agent. A value in the patient at least equal to the control valueindicates a positive response to treatment.

The invention further provides diagnostic kits for performing the abovemethods. Such kits typically include a reagent that specifically bindsto antibodies to Aβ or which stimulates proliferation of T-cellsreactive with Aβ.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Antibody titer after injection of transgenic mice with Aβ1-42.

FIG. 2: Amyloid burden in the hippocampus. The percentage of the area ofthe hippocampal region occupied by amyloid plaques, defined byreactivity with the Aβ-specific mAβ 3D6, was determined bycomputer-assisted quantitative image analysis of immunoreacted brainsections. The values for individual mice are shown sorted by treatmentgroup. The horizontal line for each grouping indicates the median valueof the distribution.

FIG. 3: Neuritic dystrophy in the hippocampus. The percentage of thearea of the hippocampal region occupied by dystrophic neurites, definedby their reactivity with the human APP-specific mAβ 8E5, was determinedby quantitative computer-assisted image analysis of immunoreacted brainsections. The values for individual mice are shown for theAN1792-treated group and the PBS-treated control group. The horizontalline for each grouping indicates the median value of the distribution.

FIG. 4: Astrocytosis in the retrosplenial cortex. The percentage of thearea of the cortical region occupied by glial fibrillary acidic protein(GFAP)-positive astrocytes was determined by quantitativecomputer-assisted image analysis of immunoreacted brain sections. Thevalues for individual mice are shown sorted by treatment group andmedian group values are indicated by horizontal lines.

FIG. 5: Geometric mean antibody titers to Aβ1-42 following immunizationwith a range of eight doses of AN1792 containing 0.14, 0.4, 1.2, 3.7,11, 33, 100, or 300 μg.

FIG. 6: Kinetics of antibody response to AN1792 immunization. Titers areexpressed as geometric means of values for the 6 animals in each group.

FIG. 7: Quantitative image analysis of the cortical amyloid burden inPBS- and AN1792-treated mice.

FIG. 8: Quantitative image analysis of the neuritic plaque burden inPBS- and AN1792-treated mice.

FIG. 9: Quantitative image analysis of the percent of the retrosplenialcortex occupied by astrocytosis in PBS- and AN1792-treated mice.

FIG. 10: Lymphocyte Proliferation Assay on spleen cells fromAN1792-treated (FIG. 10A) or PBS-treated (FIG. 10B).

FIG. 11: Total Aβ levels in the cortex. A scatterplot of individual Aβprofiles in mice immunized with Aβ or APP derivatives combined withFreund's adjuvant.

FIG. 12: Amyloid burden in the cortex was determined by quantitativeimage analysis of immunoreacted brain sections for mice immunized withthe Aβ peptide conjugates Aβ1-5, Aβ1-12, and Aβ13-28; the full length Aβaggregates AN1792 (Aβ1-42) and AN1528 (Aβ1-40) and the PBS-treatedcontrol group.

FIG. 13: Geometric mean titers of Aβ-specific antibody for groups ofmice immunized with Aβ or APP derivatives combined with Freund'sadjuvant.

FIG. 14: Geometric mean titers of Aβ-specific antibody for groups ofguinea pigs immunized with AN1792, or a palmitoylated derivativethereof, combined with various adjuvants.

FIGS. 15A-E: Aβ levels in the cortex of 12-month old PDAPP mice treatedwith AN1792 or AN1528 in combination with different adjuvants. The Aβlevel for individual mice in each treatment group, and the median, mean,and p values for each treatment group are shown.

FIG. 15A: The values for mice in the PBS-treated control group and theuntreated control group.

FIG. 15B: The values for mice in the AN1528/alum andAN1528/MPL-treatment groups.

FIG. 15C: The values for mice in the AN1528/QS21 and AN1792/Freund'sadjuvant treatment groups.

FIG. 15D: The values for mice in the AN1792/Thimerosol and AN1792/alumtreatment groups.

FIG. 15E: The values for mice in the AN1792/MPL and AN1792/QS21treatmentgroups.

DETAILED DESCRIPTION I. General

The invention provides pharmaceutical compositions and methods forprophylactic and therapeutic treatment of diseases characterized byaccumulation of amyloid deposits. Amyloid deposits comprise a peptideaggregated to an insoluble mass. The nature of the peptide varies indifferent diseases but in most cases, the aggregate has a β-pleatedsheet structure and stains with Congo Red dye. Diseases characterized byamyloid deposits include Alzheimer's disease (AD), both late and earlyonset. In both diseases, the amyloid deposit comprises a peptide termedAβ, which accumulates in the brain of affected individuals. Examples ofsome other diseases characterized by amyloid deposits are SAAamyloidosis, hereditary Icelandic syndrome, multiple myeloma, andspongiform encephalopathies, including mad cow disease, CreutzfeldtJakob disease, sheep scrapie, and mink spongiform encephalopathy (seeWeissmann et al., Curr. Opin. Neurobiol. 7, 695-700 (1997); Smits etal., Veterinary Quarterly 19, 101-105 (1997); Nathanson et al., Am. J.Epidemiol. 145, 959-969 (1997)). The peptides forming the aggregates inthese diseases are serum amyloid A, cystantin C, IgG kappa light chainrespectively for the first three, and prion protein for the others.

II. Definitions

The term “substantial identity” means that two peptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least 65 percent sequence identity, preferably atleast 80 or 90 percent sequence identity, more preferably at least 95percent sequence identity or more (e.g., 99 percent sequence identity orhigher). Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra). One example of algorithm that is suitable fordetermining percent sequence identify and sequence similarity is theBLAST algorithm, which is described in Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publiclyavailable through the National Center or Biotechnology Information(http://www.ncbi.nlm.nih.gov/). Typically, default program parameterscan be used to perform the sequence comparison, although customizedparameters can also be used. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength (W) of 3, an expectation (E) of10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc.Natl. Acad. Sci. USA 89, 10915 (1989)).

For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I(hydrophobic sidechains): norleucine, met, ala, val, leu, ile; Group H(neutral hydrophilic side chains): cys, ser, thr; Group III (acidic sidechains): asp, glu; Group IV (basic side chains): asn, gin, his, lys,arg; Group V (residues influencing chain orientation): gly, pro; andGroup VI (aromatic side chains): trp, tyr, phe. Conservativesubstitutions involve substitutions between amino acids in the sameclass. Non-conservative substitutions constitute exchanging a member ofone of these classes for a member of another.

Therapeutic agents of the invention are typically substantially pure.This means that an agent is typically at least about 50% w/w(weight/weight) purity, as well as being substantially free frominterfering proteins and contaminants. Sometimes the agents are at leastabout 80% w/w and, more preferably at least 90 or about 95% w/w purity.However, using conventional protein purification techniques, homogeneouspeptides of at least 99% w/w can be obtained.

Specific binding between two entities means an affinity of at least 10⁶,10⁷, 10⁸, 10⁹M⁻¹, or 10¹⁰ M⁻¹. Affinities greater than 10⁸ M⁻¹ arepreferred.

The term “antibody” is used to include intact antibodies and bindingfragments thereof. Typically, fragments compete with the intact antibodyfrom which they were derived for specific binding to an antigen.Optionally, antibodies or binding fragments thereof, can be chemicallyconjugated to, or expressed as, fusion proteins with other proteins.

APP⁶⁹⁵, APP⁷⁵¹, APP⁷⁷⁰ refer, respectively, to the 695, 751, and 770amino acid residue long polypeptides encoded by the human APP gene. SeeKang et al., Nature 325, 773 (1987); Ponte et al., Nature 331, 525(1988); and Kitaguchi et al., Nature 331, 530 (1988). Amino acids withinthe human amyloid precursor protein (APP) are assigned numbers accordingto the sequence of the APP770 isoform. Terms such as Aβ39, Aβ40, Aβ41,Aβ42 and Aβ43 refer to an Aβ peptide containing amino acid residues1-39, 1-40, 1-41, 1-42 and 1-43.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which B and/or T cells respond. B-cell epitopes can be formedboth from contiguous amino acids or noncontiguous amino acids juxtaposedby tertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). Antibodies thatrecognize the same epitope can be identified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen. T-cells recognize continuous epitopes ofabout nine amino acids for CD8 cells or about 13-15 amino acids for CD4cells. T cells that recognize the epitope can be identified by in vitroassays that measure antigen-dependent proliferation, as determined by³H-thymidine incorporation by primed T cells in response to an epitope(Burke et al., J. Inf. Dis. 170, 1110-19 (1994)), by antigen-dependentkilling (cytotoxic T lymphocyte assay, Tigges et al., J. Immunol. 156,3901-3910) or by cytokine secretion.

The term “immunological” or “immune” response is the development of abeneficial humoral (antibody mediated) and/or a cellular (mediated byantigen-specific T cells or their secretion products) response directedagainst an amyloid peptide in a recipient patient. Such a response canbe an active response induced by administration of immunogen or apassive response induced by administration of antibody or primedT-cells. A cellular immune response is elicited by the presentation ofpolypeptide epitopes in association with Class I or Class II MHCmolecules to activate antigen-specific CD4⁺ T helper cells and/or CD8⁺cytotoxic T cells. The response may also involve activation ofmonocytes, macrophages, NK cells, basophils, dendritic cells,astrocytes, microglia cells, eosinophils or other components of innateimmunity. The presence of a cell-mediated immunological response can bedetermined by proliferation assays (CD4⁺ T cells) or CTL (cytotoxic Tlymphocyte) assays (see Burke, supra; Tigges, supra). The relativecontributions of humoral and cellular responses to the protective ortherapeutic effect of an immunogen can be distinguished by separatelyisolating IgG and T-cells from an immunized syngeneic animal andmeasuring protective or therapeutic effect in a second subject.

An “immunogenic agent” or “immunogen” is capable of inducing animmunological response against itself on administration to a patient,optionally in conjunction with an adjuvant.

The term “naked polynucleotide” refers to a polynucleotide not complexedwith colloidal materials. Naked polynucleotides are sometimes cloned ina plasmid vector.

The term “adjuvant” refers to a compound that when administered inconjunction with an antigen augments the immune response to the antigen,but when administered alone does not generate an immune response to theantigen. Adjuvants can augment an immune response by several mechanismsincluding lymphocyte recruitment, stimulation of B and/or T cells, andstimulation of macrophages.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

Disaggregated or monomeric Aβ means soluble, monomeric peptide units ofAβ. One method to prepare monomeric Aβ is to dissolve lyophilizedpeptide in neat DMSO with sonication. The resulting solution iscentrifuged to remove any nonsoluble particulates. Aggregated Aβ is amixture of oligomers in which the monomeric units are held together bynoncovalent bonds.

Compositions or methods “comprising” one or more recited elements mayinclude other elements not specifically recited. For example, acomposition that comprises Aβ peptide encompasses both an isolated Aβpeptide and Aβ peptide as a component of a larger polypeptide sequence.

Therapeutic Agents

1. Alzheimer's Disease

Therapeutic agents for use in the present invention induce an immuneresponse against Aβ peptide. These agents include Aβ peptide itself andvariants thereof, analogs and mimetics of Aβ peptide that induce and/orcrossreact with antibodies to Aβ peptide, and antibodies or T-cellsreactive with Aβ peptide. Induction of an immune response can be activeas when an immunogen is administered to induce antibodies or T-cellsreactive with Aβ in a patient, or passive, as when an antibody isadministered that itself binds to Aβ in patient.

Aβ, also known as β-amyloid peptide, or A4 peptide (see U.S. Pat. No.4,666,829; Glenner & Wong, Biochem. Biophys. Res. Commun. 120, 1131(1984)), is a peptide of 39-43 amino acids, which is the principalcomponent of characteristic plaques of Alzheimer's disease. Aβ isgenerated by processing of a larger protein APP by two enzymes, termed βand γ secretases (see Hardy, TINS 20, 154 (1997)). Known mutations inAPP associated with Alzheimer's disease occur proximate to the site of βor γ secretase, or within Aβ. For example, position 717 is proximate tothe site of γ-secretase cleavage of APP in its processing to Aβ, andpositions 670/671 are proximate to the site of β-secretase cleavage. Itis believed that the mutations cause AD disease by interacting with thecleavage reactions by which Aβ is formed so as to increase the amount ofthe 42/43 amino acid form of Aβ generated.

Aβ has the unusual property that it can fix and activate both classicaland alternate complement cascades. In particular, it binds to Clq andultimately to C3bi. This association facilitates binding to macrophagesleading to activation of B cells. In addition, C3bi breaks down furtherand then binds to CR2 on B cells in a T cell dependent manner leading toa 10,000 increase in activation of these cells. This mechanism causes Aβto generate an immune response in excess of that of other antigens.

The therapeutic agent used in the claimed methods can be any of thenaturally occurring forms of Aβ peptide, and particularly the humanforms (i.e., Aβ39, Aβ40, Aβ41, Aβ or Aβ43). The sequences of thesepeptides and their relationship to the APP precursor are illustrated byFIG. 1 of Hardy et al., TINS 20, 155-158 (1997). For example, Aβ has thesequence:

-   -   H₂N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala-OH        (SEQ ID NO: 1).

Aβ41, Aβ40 and Aβ39 differ from Aβ42 by the omission of Ala, Ala-Ile,and Ala-Ile-Val respectively from the C-terminal end. Aβ43 differs fromAβ42 by the presence of a threonine residue at the C-terminus. Thetherapeutic agent can also be an active fragment or analog of a naturalAβ peptide that contains an epitope that induces a similar protective ortherapeutic immune response on administration to a human. Immunogenicfragments typically have a sequence of at least 3, 5, 6, 1 0 or 20contiguous amino acids from a natural peptide. Immunogenic fragmentsinclude Aβ1-5, 1-6, 1-12, 13-28, 17-28, 25-25, 35-40 and 35-42.Fragments from the N-terminal half of Aβ are preferred in some methods.Analogs include allelic, species and induced variants. Analogs typicallydiffer from naturally occurring peptides at one or a few positions,often by virtue of conservative substitutions. Analogs typically exhibitat least 80 or 90% sequence identity with natural peptides. Some analogsalso include unnatural amino acids or modifications of N or C terminalamino acids. Examples of unnatural amino acids are α,α-disubstitutedamino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, ω-N-methylarginine. Fragments and analogs can bescreened for prophylactic or therapeutic efficacy in transgenic animalmodels as described below.

Aβ, its fragments, analogs and other amyloidogenic peptides can besynthesized by solid phase peptide synthesis or recombinant expression,or can be obtained from natural sources. Automatic peptide synthesizersare commercially available from numerous suppliers, such as AppliedBiosystems, Foster City, Calif. Recombinant expression can be inbacteria, such as E. coli, yeast, insect cells or mammalian cells.Procedures for recombinant expression are described by Sambrook et al.,Molecular Cloning: A Laboratory Manual (C.S.H.P. Press, NY 2d ed.,1989). Some forms of Aβ peptide are also available commercially (e.g.,American Peptides Company, Inc., Sunnyvale, Calif. and CaliforniaPeptide Research, Inc. Napa, Calif.).

Therapeutic agents also include longer polypeptides that include, forexample, an Aβ peptide, active fragment or analog together with otheramino acids. For example, Aβ peptide can be present as intact APPprotein or a segment thereof, such as the C-100 fragment that begins atthe N-terminus of Aβ and continues to the end of APP. Such polypeptidescan be screened for prophylactic or therapeutic efficacy in animalmodels as described below. The Aβ peptide, analog, active fragment orother polypeptide can be administered in associated form (i.e., as anamyloid peptide) or in dissociated form. Therapeutic agents also includemultimers of monomeric immunogenic agents.

In a further variation, an immunogenic peptide, such as Aβ, can bepresented as a viral or bacterial vaccine. A nucleic acid encoding theimmunogenic peptide is incorporated into a genome or episome of thevirus or bacteria. Optionally, the nucleic acid is incorporated in sucha manner that the immunogenic peptide is expressed as a secreted proteinor as a fusion protein with an outersurface protein of a virus or atransmembrane protein of a bacteria so that the peptide is displayed.Viruses or bacteria used in such methods should be nonpathogenic orattenuated. Suitable viruses include adenovirus, HSV, vaccinia and fowlpox. Fusion of an immunogenic peptide to HBsAg of HBV is particularlysuitable. Therapeutic agents also include peptides and other compoundsthat do not necessarily have a significant amino acid sequencesimilarity with Aβ but nevertheless serve as mimetics of Aβ and induce asimilar immune response. For example, any peptides and proteins formingβ-pleated sheets can be screened for suitability. Anti-idiotypicantibodies against monoclonal antibodies to Aβ or other amyloidogenicpeptides can also be used. Such anti-Id antibodies mimic the antigen andgenerate an immune response to it (see Essential Immunology (Roit ed.,Blackwell Scientific Publications, Palo Alto, 6th ed.), p. 181).

Random libraries of peptides or other compounds can also be screened forsuitability. Combinatorial libraries can be produced for many types ofcompounds that can be synthesized in a step-by-step fashion. Suchcompounds include polypeptides, beta-turn mimetics, polysaccharides,phospholipids, hormones, prostaglandins, steroids, aromatic compounds,heterocyclic compounds, benzodiazepines, oligomeric N-substitutedglycines and oligocarbamates. Large combinatorial libraries of thecompounds can be constructed by the encoded synthetic libraries (ESL)method described in Affymax, WO 95/12608, Affymax, WO 93/06121, ColumbiaUniversity, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO95/30642 (each of which is incorporated by reference for all purposes).Peptide libraries can also be generated by phage display methods. See,e.g., Devlin, WO 91/18980.

Combinatorial libraries and other compounds are initially screened forsuitability by determining their capacity to bind to antibodies orlymphocytes (B or T) known to be specific for Aβ or other amyloidogenicpeptides. For example, initial screens can be performed with anypolyclonal sera or monoclonal antibody to Aβ or other amyloidogenicpeptide. Compounds identified by such screens are then further analyzedfor capacity to induce antibodies or reactive lymphocytes to Aβ or otheramyloidogenic peptide. For example, multiple dilutions of sera can betested on microtiter plates that have been precoated with Aβ peptide anda standard ELISA can be performed to test for reactive antibodies to Aβ.Compounds can then be tested for prophylactic and therapeutic efficacyin transgenic animals predisposed to an amyloidogenic disease, asdescribed in the Examples. Such animals include, for example, micebearing a 717 mutation of APP described by Games et al., supra, and micebearing a Swedish mutation of APP such as described by McConlogue etal., U.S. Pat. No. 5,612,486 and Hsiao et al., Science 274, 99 (1996);Staufenbiel et al., Proc. Natl. Acad. Sci. USA 94, 13287-13292 (1997);Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. USA 94, 13287-13292(1997); Borchelt et al., Neuron 19, 939-945 (1997)). The same screeningapproach can be used on other potential agents such as fragments of Aβ,analogs of Aβ and longer peptides including Aβ, described above.

Therapeutic agents of the invention also include antibodies thatspecifically bind to Aβ. Such antibodies can be monoclonal orpolyclonal. Some such antibodies bind specifically to the aggregatedform of Aβ without binding to the dissociated form. Some bindspecifically to the dissociated form without binding to the aggregatedform. Some bind to both aggregated and dissociated forms. The productionof non-human monoclonal antibodies, e.g., murine or rat, can beaccomplished by, for example, immunizing the animal with Aβ. See Harlow& Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated byreference for all purposes). Such an immunogen can be obtained from anatural source, by peptides synthesis or by recombinant expression.

Humanized forms of mouse antibodies can be generated by linking the CDRregions of non-human antibodies to human constant regions by recombinantDNA techniques. See Queen et al., Proc. Natl. Acad Sci. USA 86,10029-10033 (1989) and WO 90/07861 (incorporated by reference for allpurposes).

Human antibodies can be obtained using phage-display methods. See, e.g.,Dower et al., WO 91/17271; McCafferty et al., WO 92/01047. In thesemethods, libraries of phage are produced in which members displaydifferent antibodies on their outersurfaces. Antibodies are usuallydisplayed as Fv or Fab fragments. Phage displaying antibodies with adesired specificity are selected by affinity enrichment to Aβ, orfragments thereof. Human antibodies against Aβ can also be produced fromnon-human transgenic mammals having transgenes encoding at least asegment of the human immunoglobulin locus and an inactivated endogenousimmunoglobulin locus. See, e.g., Lonberg et al., WO93/12227 (1993);Kucherlapati, WO 91/10741 (1991) (each of which is incorporated byreference in its entirety for all purposes). Human antibodies can beselected by competitive binding experiments, or otherwise, to have thesame epitope specificity as a particular mouse antibody. Such antibodiesare particularly likely to share the useful functional properties of themouse antibodies. Human polyclonal antibodies can also be provided inthe form of serum from humans immunized with an immunogenic agent.Optionally, such polyclonal antibodies can be concentrated by affinitypurification using Aβ or other amyloid peptide as an affinity reagent.

Human or humanized antibodies can be designed to have IgG, IgD, IgA andIgE constant region, and any isotype, including IgG1, IgG2, IgG3 andIgG4. Antibodies can be expressed as tetramers containing two light andtwo heavy chains, as separate heavy chains, light chains, as Fab, Fab′F(ab′)₂, and Fv, or as single chain antibodies in which heavy and lightchain variable domains are linked through a spacer.

Therapeutic agents for use in the present methods also include T-cellsthat bind to Aβ peptide. For example, T-cells can be activated againstAβ peptide by expressing a human MHC class I gene and a humanβ-2-microglobulin gene from an insect cell line, whereby an emptycomplex is formed on the surface of the cells and can bind to Aβpeptide. T-cells contacted with the cell line become specificallyactivated against the peptide. See Peterson et al., U.S. Pat. No.5,314,813. Insect cell lines expressing an MHC class II antigen cansimilarly be used to activate CD4 T cells.

Other Diseases

The same or analogous principles determine production of therapeuticagents for treatment of other amyloidogenic diseases. In general, theagents noted above for use in treatment of Alzheimer's disease can alsobe used for treatment early onset Alzheimer's disease associated withDown's syndrome. In mad cow disease, prion peptide, active fragments,and analogs, and antibodies to prion peptide are used in place of Aβpeptide, active fragments, analogs and antibodies to Aβ peptide intreatment of Alzheimer's disease. In treatment of multiple myeloma, IgGlight chain and analogs and antibodies thereto are used, and so forth inother diseases.

3. Carrier Proteins

Some agents for inducing an immune response contain the appropriateepitope for inducing an immune response against amyloid deposits but aretoo small to be immunogenic. In this situation, a peptide immunogen canbe linked to a suitable carrier to help elicit an immune response.Suitable carriers include serum albumins, keyhole limpet hemocyanin,immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, or atoxoid from other pathogenic bacteria, such as diphtheria, E. coli,cholera, or H. pylori, or an attenuated toxin derivative. Other carriersfor stimulating or enhancing an immune response include cytokines suchas IL-1, IL-1α and β peptides, IL-2, γINF, IL-10, GM-CSF, andchemokines, such as M1P1α and β and RANTES. Immunogenic agents can alsobe linked to peptides that enhance transport across tissues, asdescribed in O'Mahony, WO 97/17613 and WO 97/17614.

Immunogenic agents can be linked to carriers by chemical crosslinking.Techniques for linking an immunogen to a carrier include the formationof disulfide linkages using N-succinimidyl-3-(2-pyridyl-thio)propionate(SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC) (if the peptide lacks a sulfhydryl group, this can be provided byaddition of a cysteine residue). These reagents create a disulfidelinkage between themselves and peptide cysteine resides on one proteinand an amide linkage through the ε-amino on a lysine, or other freeamino group in other amino acids. A variety of suchdisulfide/amide-forming agents are described by Immun. Rev. 62, 185(1982). Other bifunctional coupling agents form a thioether rather thana disulfide linkage. Many of these thio-ether-forming agents arecommercially available and include reactive esters of 6-maleimidocaproicacid, 2-bromoacetic acid, and 2-iodoacetic acid,4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid. The carboxyl groupscan be activated by combining them with succinimide or1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.

Immunogenic peptides can also be expressed as fusion proteins withcarriers. The immunogenic peptide can be linked at the amino terminus,the carboxyl terminus, or internally to the carrier. Optionally,multiple repeats of the immunogenic peptide can be present in the fusionprotein.

4. Nucleic Acid Encoding Immunogens

Immune responses against amyloid deposits can also be induced byadministration of nucleic acids encoding Aβ peptide or other peptideimmunogens. Such nucleic acids can be DNA or RNA. A nucleic acid segmentencoding the immunogen is typically linked to regulatory elements, suchas a promoter and enhancer, that allow expression of the DNA segment inthe intended target cells of a patient. For expression in blood cells,as is desirable for induction of an immune response, promoter andenhancer elements from light or heavy chain immunoglobulin genes or theCMV major intermediate early promoter and enhancer are suitable todirect expression. The linked regulatory elements and coding sequencesare often cloned into a vector.

A number of viral vector systems are available including retroviralsystems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3,102-109 (1993)); adenoviral vectors (see, e.g., Bett et al., J. Virol.67, 5911 (1993)); adeno-associated virus vectors (see, e.g., Zhou etal., J. Exp. Med. 179, 1867 (1994)), viral vectors from the pox familyincluding vaccinia virus and the avian pox viruses, viral vectors fromthe alpha virus genus such as those derived from Sindbis and SemlikiForest Viruses (see, e.g., Dubensky et al., J. Virol. 70, 508-519(1996)), and papillomaviruses (Ohe et al., Human Gene Therapy 6, 325-333(1995); Woo et al., WO 94/12629 and Xiao & Brandsma, Nucleic Acids. Res.24, 2630-2622 (1996)).

DNA encoding an immunogen, or a vector containing the same, can bepackaged into liposomes. Suitable lipids and related analogs aredescribed by U.S. Pat. Nos. 5,208,036, 5,264,618, 5,279,833 and5,283,185. Vectors and DNA encoding an immunogen can also be adsorbed toor associated with particulate carriers, examples of which includepolymethyl methacrylate polymers and polylactides andpoly(lactide-co-glycolides), see, e.g., McGee et al., J. Micro Encap.(1996).

Gene therapy vectors or naked DNA can be delivered in vivo byadministration to an individual patient, typically by systemicadministration (e.g., intravenous, intraperitoneal, nasal, gastric,intradermal, intramuscular, subdermal, or intracranial infusion) ortopical application (see e.g., U.S. Pat. No. 5,399,346). DNA can also beadministered using a gene gun. See Xiao & Brandsma, supra. The DNAencoding an immunogen is precipitated onto the surface of microscopicmetal beads. The microprojectiles are accelerated with a shock wave orexpanding helium gas, and penetrate tissues to a depth of several celllayers. For example, The Accel™ Gene Delivery Device manufactured byAgacetus, Inc. Middleton, Wis. is suitable. Alternatively, naked DNA canpass through skin into the blood stream simply by spotting the DNA ontoskin with chemical or mechanical irritation (see WO 95/05853).

In a further variation, vectors encoding immunogens can be delivered tocells ex vivo, such as cells explanted from an individual patient (e.g.,lymphocytes, bone marrow aspirates, tissue biopsy) or universal donorhematopoietic stem cells, followed by reimplantation of the cells into apatient, usually after selection for cells which have incorporated thevector.

IV. Patients Amenable to Treatment

Patients amenable to treatment include individuals at risk of diseasebut not showing symptoms, as well as patients presently showingsymptoms. In the case of Alzheimer's disease, virtually anyone is atrisk of suffering from Alzheimer's disease if he or she lives longenough. Therefore, the present methods can be administeredprophylactically to the general population without any assessment of therisk of the subject patient. The present methods are especially usefulfor individuals who do have a known genetic risk of Alzheimer's disease.Such individuals include those having relatives who have experiencedthis disease, and those whose risk is determined by analysis of geneticor biochemical markers. Genetic markers of risk toward Alzheimer'sdisease include mutations in the APP gene, particularly mutations atposition 717 and positions 670 and 671 referred to as the Hardy andSwedish mutations respectively (see Hardy, TINS, supra). Other markersof risk are mutations in the presenilin genes, PS1 and PS2, and ApoE4,family history of AD, hypercholesterolemia or atherosclerosis.Individuals presently suffering from Alzheimer's disease can berecognized from characteristic dementia, as well as the presence of riskfactors described above. In addition, a number of diagnostic tests areavailable for identifying individuals who have AD. These includemeasurement of CSF tau and Aβ42 levels. Elevated tau and decreased Aβ42levels signify the presence of AD. Individuals suffering fromAlzheimer's disease can also be diagnosed by MMSE or ADRDA criteria asdiscussed in the Examples section.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70. Treatment typically entails multipledosages over a period of time. Treatment can be monitored by assayingantibody, or activated T-cell or B-cell responses to the therapeuticagent (e.g., Aβ peptide) over time. If the response falls, a boosterdosage is indicated. In the case of potential Down's syndrome patients,treatment can begin antenatally by administering therapeutic agent tothe mother or shortly after birth.

V. Treatment Regimes

In prophylactic applications, pharmaceutical compositions or medicantsare administered to a patient susceptible to, or otherwise at risk of, aparticular disease in an amount sufficient to eliminate or reduce therisk or delay the outset of the disease. In therapeutic applications,compositions or medicants are administered to a patient suspected of, oralready suffering from such a disease in an amount sufficient to cure,or at least partially arrest, the symptoms of the disease and itscomplications. An amount adequate to accomplish this is defined as atherapeutically- or pharmaceutically-effective dose. In bothprophylactic and therapeutic regimes, agents are usually administered inseveral dosages until a sufficient immune response has been achieved.Typically, the immune response is monitored and repeated dosages aregiven if the immune response starts to fade.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human, but insome diseases, such as mad cow disease, the patient can be a nonhumanmammal, such as a bovine. Treatment dosages need to be titrated tooptimize safety and efficacy. The amount of immunogen depends on whetheradjuvant is also administered, with higher dosages being required in theabsence of adjuvant. The amount of an immunogen for administrationsometimes varies from 1 μg-500 μg per patient and more usually from5-500 μg per injection for human administration. Occasionally, a higherdose of 1-2 mg per injection is used. Typically about 10, 20, 50 or 100μg is used for each human injection. The timing of injections can varysignificantly from once a day, to once a year, to once a decade. On anygiven day that a dosage of immunogen is given, the dosage is greaterthan 1 μg/patient and usually greater than 10 μg/patient if adjuvant isalso administered, and greater than 10 μg/patient and usually greaterthan 100 μg/patient in the absence of adjuvant. A typical regimenconsists of an immunization followed by booster injections at 6 weeklyintervals. Another regimen consists of an immunization followed bybooster injections 1, 2 and 12 months later. Another regimen entails aninjection every two months for life. Alternatively, booster injectionscan be on an irregular basis as indicated by monitoring of immuneresponse. For passive immunization with an antibody, the dosage rangesfrom about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg of thehost body weight. Doses for nucleic acids encoding immunogens range fromabout 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNAper patient. Doses for infectious viral vectors vary from 10-10⁹, ormore, virions per dose.

Agents for inducing an immune response can be administered byparenteral, topical, intravenous, oral, subcutaneous, intraperitoneal,intranasal or intramuscular means for prophylactic and/or therapeutictreatment. The most typical route of administration is subcutaneousalthough others can be equally effective. The next most common isintramuscular injection. This type of injection is most typicallyperformed in the arm or leg muscles. Intravenous injections as well asintraperitoneal injections, intraarterial, intracranial, or intradermalinjections are also effective in generating an immune response. In somemethods, agents are injected directly into a particular tissue wheredeposits have accumulated.

Agents of the invention can optionally be administered in combinationwith other agents that are at least partly effective in treatment ofamyloidogenic disease. In the case of Alzheimer's and Down's syndrome,in which amyloid deposits occur in the brain, agents of the inventioncan also be administered in conjunction with other agents that increasepassage of the agents of the invention across the blood-brain barrier.

Immunogenic agents of the invention, such as peptides, are sometimesadministered in combination with an adjuvant. A variety of adjuvants canbe used in combination with a peptide, such as AD, to elicit an immuneresponse. Preferred adjuvants augment the intrinsic response to animmunogen without causing conformational changes in the immunogen thataffect the qualitative form of the response. Preferred adjuvants includealum, 3 De-O-acylated monophosphoryl lipid A (MPL) (see GB 2220211).QS21 is a triterpene glycoside or saponin isolated from the bark of theQuillaja Saponaria Molina tree found in South America (see Kensil etal., in Vaccine Design: The Subunit and Ajuvant Approach (eds. Powell &Newman, Plenum Press, NY, 1995); U.S. Pat. No. 5,057,540). Otheradjuvants are oil in water emulsions (such as squalene or peanut oil),optionally in combination with immune stimulants, such as monophosphoryllipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)). Anotheradjuvant is CpG (Bioworld Today, Nov. 15, 1998). Alternatively, Aβ canbe coupled to an adjuvant. For example, a lipopeptide version of Aβ canbe prepared by coupling palmitic acid or other lipids directly to theN-terminus of Aβ as described for hepatitis B antigen vaccination(Livingston, J. Immunol. 159, 1383-1392 (1997)). However, such couplingshould not substantially change the conformation of Aβ so as to affectthe nature of the immune response thereto. Adjuvants can be administeredas a component of a therapeutic composition with an active agent or canbe administered separately, before, concurrently with, or afteradministration of the therapeutic agent.

A preferred class of adjuvants is aluminum salts (alum), such asaluminum hydroxide, aluminum phosphate, aluminum sulfate. Such adjuvantscan be used with or without other specific immunostimulating agents suchas MPL or 3-DMP, QS21, polymeric or monomeric amino acids such aspolyglutamic acid or polylysine. Another class of adjuvants isoil-in-water emulsion formulations. Such adjuvants can be used with orwithout other specific immunostimulating agents such as muramyl peptides(e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-A1-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) theramide™), or other bacterial cell wallcomponents. Oil-in-water emulsions include (a) MF59 (WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton Mass.), (b) SAF, containing 10% Squalane, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, and (c) Ribi™ adjuvant system (RAS),(Ribi Immunochem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween80, and one or more bacterial cell wall components from the groupconsisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (Detox™). Another classof preferred adjuvants is saponin adjuvants, such as Stimulon™ (QS21,Aquila, Worcester, Mass.) or particles generated therefrom such asISCOMs (immunostimulating complexes) and ISCOMATRIX. Other adjuvantsinclude Complete Freund's Adjuvant (CFA) and Incomplete Freund'sAdjuvant (IFA). Other adjuvants include cytokines, such as interleukins(IL-1, IL-2, and IL-12), macrophage colony stimulating factor (M-CSF),tumor necrosis factor (TNF).

An adjuvant can be administered with an immunogen as a singlecomposition, or can be administered before, concurrent with or afteradministration of the immunogen. Immunogen and adjuvant can be packagedand supplied in the same vial or can be packaged in separate vials andmixed before use. Immunogen and adjuvant are typically packaged with alabel indicating the intended therapeutic application. If immunogen andadjuvant are packaged separately, the packaging typically includesinstructions for mixing before use. The choice of an adjuvant and/orcarrier depends on the stability of the vaccine containing the adjuvant,the route of administration, the dosing schedule, the efficacy of theadjuvant for the species being vaccinated, and, in humans, apharmaceutically acceptable adjuvant is one that has been approved or isapprovable for human administration by pertinent regulatory bodies. Forexample, Complete Freund's adjuvant is not suitable for humanadministration. Alum, MPL and QS21 are preferred. Optionally, two ormore different adjuvants can be used simultaneously. Preferredcombinations include alum with MPL, alum with QS21, MPL with QS21, andalum, QS21 and MPL together. Also, Incomplete Freund's adjuvant can beused (Chang et al., Advanced Drug Delivery Reviews 32, 173-186 (1998)),optionally in combination with any of alum, QS21, and MPL and allcombinations thereof.

Agents of the invention are often administered as pharmaceuticalcompositions comprising an active therapeutic agent, i.e., and a varietyof other pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.,1980). The preferred form depends on the intended mode of administrationand therapeutic application. The compositions can also include,depending on the formulation desired, pharmaceutically-acceptable,non-toxic carriers or diluents, which are defined as vehicles commonlyused to formulate pharmaceutical compositions for animal or humanadministration. The diluent is selected so as not to affect thebiological activity of the combination. Examples of such diluents aredistilled water, physiological phosphate-buffered saline, Ringer'ssolutions, dextrose solution, and Hank's solution. In addition, thepharmaceutical composition or formulation may also include othercarriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like. However, some reagents suitable foradministration to animals, such as Complete Freund's adjuvant are nottypically included in compositions for human use.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides, polylactic acids,polyglycolic acids and copolymers (such as latex functionalizedsepharose, agarose, cellulose, and the like), polymeric amino acids,amino acid copolymers, and lipid aggregates (such as oil droplets orliposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

For parenteral administration, agents of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier which can be a sterile liquid such as water oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable f6r solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above (see Langer, Science 249,1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28, 97-119 (1997).The agents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications.

For suppositories, binders and carriers include, for example,polyalkylene glycols or triglycerides; such suppositories can be formedfrom mixtures containing the active ingredient in the range of 0.5% to10%, preferably 1%-2%. Oral formulations include excipients, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, and magnesium carbonate. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10%-95%of active ingredient, preferably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (See Glenn et al., Nature 391, 851(1998)). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin path orusing transferosomes (Paul et al., Eur. J. Immunol. 25, 3521-24 (1995);Cevc et al., Biochem. Biophys. Acta 1368, 201-15 (1998)).

VI. Methods of Diagnosis

The invention provides methods of detecting an immune response againstAβ peptide in a patient suffering from or susceptible to Alzheimer'sdisease. The methods are particularly useful for monitoring a course oftreatment being administered to a patient. The methods can be used tomonitor both therapeutic treatment on symptomatic patients andprophylactic treatment on asymptomatic patients.

Some methods entail determining a baseline value of an immune responsein a patient before administering a dosage of agent, and comparing thiswith a value for the immune response after treatment. A significantincrease (i.e., greater than the typical margin of experimental error inrepeat measurements of the same sample, expressed as one standarddeviation from the mean of such measurements) in value of the immuneresponse signals a positive treatment outcome (i.e., that administrationof the agent has achieved or augmented an immune response). If the valuefor immune response does not change significantly, or decreases, anegative treatment outcome is indicated. In general, patients undergoingan initial course of treatment with an agent are expected to show anincrease in immune response with successive dosages, which eventuallyreaches the plateau. Administration of agent is generally continuedwhile the immune response is increasing. Attainment of the plateau is anindicator that the administered of treatment can be discontinued orreduced in dosage or frequency.

In other methods, a control value (i.e., a mean and standard deviation)of immune response is determined for a control population. Typically theindividuals in the control population have not received prior treatment.Measured values of immune response in a patient after administering atherapeutic agent are then compared with the control value. Asignificant increase relative to the control value (e.g., greater thanone standard deviation from the mean) signals a positive treatmentoutcome. A lack of significant increase or a decrease signals a negativetreatment outcome. Administration of agent is generally continued whilethe immune response is increasing relative to the control value. Asbefore, attainment of a plateau relative to control values in anindicator that the administration of treatment can be discontinued orreduced in dosage or frequency.

In other methods, a control value of immune response (e.g., a mean andone standard deviation) is determined from a control population ofindividuals who have undergone treatment with a therapeutic agent andwhose immune responses have plateaued in response to treatment. Measuredvalues of immune response in a patient are compared with the controlvalue. If the measured level in a patient is not significantly different(e.g., more than one standard deviation) from the control value,treatment can be discontinued. If the level in a patient issignificantly below the control value, continued administration of agentis warranted. If the levels in the patient persist below the controlvalue, then a change in treatment regime, for example, use of adifferent adjuvant may be indicated.

In other methods, a patient who is not presently receiving treatment buthas undergone a previous course of treatment is monitored for immuneresponse to determine whether a resumption of treatment is required. Themeasured value of immune response in the patient can be compared with avalue of immune response previously achieved in the patient after aprevious course of treatment. A significant decrease relative to theprevious measurement (i.e., greater than a typical margin of error inrepeat measurements of the same value) is an indication that treatmentcan be resumed. Alternatively, the value measured in patient can becompared with a control value (mean plus standard deviation) determinedin population of patients after undergoing a course of treatment.Alternatively, the measured value in a patient can be compared with acontrol value in populations of prophylactically treated patients whoremain free of symptoms of disease, or populations of therapeuticallytreated patients who show amelioration of disease characteristics. Inall of these cases, a significant decrease relative to the control level(i.e., more than a standard deviation) is an indicator that treatmentshould be resumed in a patient.

The tissue sample for analysis is typically blood, plasma, serum, mucusor cerebral spinal fluid from the patient. The sample is analyzed forindicia of an immune response to any forms of Aβ peptide, typicallyAβ42. The immune response can be determined from the presence of, e.g.,antibodies or T-cells that specifically bind to Aβ peptide. ELISAmethods of detecting antibodies specific to Aβ are described in theExamples section. Methods of detecting reactive T-cells have beendescribed above (see Definitions).

The invention further provides diagnostic kits for performing thediagnostic methods described above. Typically, such kits contain anagent that specifically binds to antibodies to Aβ or reacts with T-cellsspecific for Aβ. The kit can also include a label. For detection ofantibodies to Aβ, the label is typically in the form of labelledanti-idiotypic antibodies. For detection of antibodies, the agent can besupplied prebound to a solid phase, such as to the wells of a microtiterdish. For detection of reactive T-cells, the label can be supplied as³H-thymidine to measure a proliferative response. Kits also typicallycontain labelling providing directions for use of the kit. The labellingmay also include a chart or other correspondence regime correlatinglevels of measured label with levels of antibodies to Aβ or T-cellsreactive with Aβ. The term labelling refers to any written or recordedmaterial that is attached to, or otherwise accompanies a kit at any timeduring its manufacture, transport, sale or use. For example, the termlabelling encompasses advertising leaflets and brochures, packagingmaterials, instructions, audio or video cassettes, computer discs, aswell as writing imprinted directly on kits.

EXAMPLE I Prophylactic Efficacy of Aβ Against AD

These examples describe administration of Aβ42 peptide to transgenicmice overexpressing APP with a mutation at position 717 (APP_(717V→F))that predisposes them to develop Alzheimer's-like neuropathology.Production and characteristics of these mice (PDAPP mice) is describedin Games et al., Nature, supra. These animals, in their heterozygoteform, begin to deposit Aβ at six months of age forward. By fifteenmonths of age they exhibit levels of Aβ deposition equivalent to thatseen in Alzheimer's disease. PDAPP mice were injected with aggregatedAβ₄₂ (aggregated Aβ₄₂) or phosphate buffered saline. Aggregated Aβ₄₂ waschosen because of its ability to induce antibodies to multiple epitopesof Aβ.

A. Methods

1. Source of Mice

Thirty PDAPP heterogenic female mice were randomly divided into thefollowing groups: 10 mice to be injected with aggregated Aβ₄₂ (one diedin transit), 5 mice to be injected with PBS/adjuvant or PBS, and 10uninjected controls. Five mice were injected with serum amyloid protein(SAP).

2. Preparation of Immunogens

Preparation of aggregated Aβ₄₂: two milligrams of Aβ₄₂ (US Peptides Inc,lot K-42-12) was dissolved in 0.9 ml water and made up to 1 ml by adding0.1 ml 10×PBS. This was vortexed and allowed to incubate overnight 37°C., under which conditions the peptide aggregated. Any unused Aβ wasstored as a dry lyophilized powder at −20° C. until the next injection.

3. Preparation of Injections

100 μg of aggregated Aβ₄₂ in PBS per mouse was emulsified 1:1 withComplete Freund's adjuvant (CFA) in a final volume of 400 μl emulsionfor the first immunization, followed by a boost of the same amount ofimmunogen in Incomplete Freund's adjuvant (IFA) at 2 weeks. Twoadditional doses in IFA were given at monthly intervals. The subsequentimmunizations were done at monthly intervals in 500 μl of PBS.Injections were delivered intraperitoneally (i.p.).

PBS injections followed the same schedule and mice were injected with a1:1 mix of PBS/Adjuvant at 400 μl per mouse, or 500 μl of PBS per mouse.SAP injections likewise followed the same schedule using a dose of 100μg per injection.

4. Titration of Mouse Bleeds, Tissue Preparation andImmunohistochemistry

B. Results

PDAPP mice were injected with either aggregated Aβ₄₂ (aggregated Aβ₄₂),SAP peptides, or phosphate buffered saline. A group of PDAPP mice werealso left as uninjected, positive controls. The titers of the mice toaggregated Aβ₄₂ were monitored every other month from the fourth boostuntil the mice were one year of age. Mice were sacrificed at 13 months.At all time points examined, eight of the nine aggregated Aβ₄₂ micedeveloped a high antibody titer, which remained high throughout theseries of injections (titers greater than 1/10000). The ninth mouse hada low, but measurable titer of approximately 1/1000 (FIG. 1, Table 1).SAPP-injected mice had titers of 1:1,000 to 1:30,000 for this immunogenwith only a single mice exceeding 1:10,0000.

The PBS-treated mice were titered against aggregated Aβ₄₂ at six, tenand twelve months. At a 1/100 dilution the PBS mice when titered againstaggregated Aβ₄₂ only exceeded 4 times background at one data point,otherwise, they were less than 4 times background at all time points(Table 1). The SAP-specific response was negligible at these time pointswith all titers less than 300.

Seven out of the nine mice in the aggregated Aβ1-42 group had nodetectable amyloid in their brains. In contrast, brain tissue from micein the SAP and PBS groups contained numerous 3D6-positive amyloiddeposits in the hippocampus, as well as in the frontal and cingulatecortices. The pattern of deposition was similar to that of untreatedcontrols, with characteristic involvement of vulnerable subregions, suchas the outer molecular layer of the hippocampal dentate gyrus. One mousefrom the Aβ1-42-injected group had a greatly reduced amyloid burden,confined to the hippocampus. An isolated plaque was identified inanother Aβ1-42-treated mouse.

Quantitative image analyses of the amyloid burden in the hippocampusverified the dramatic reduction achieved in the AN1792-treated animals(FIG. 2). The median values of the amyloid burden for the PBS group(2.22%), and for the untreated control group (2.65%) were significantlygreater than for those immunized with AN1792 (0.00%, p=0.0005). Incontrast, the median value for the group immunized with SAP peptides(SAPP) was 5.74%. Brain tissue from the untreated, control micecontained numerous Aβ amyloid deposits visualized with the Aβ-specificmonoclonal antibody (mAβ) 3D6 in the hippocampus, as well as in theretrosplenial cortex. A similar pattern of amyloid deposition was alsoseen in mice immunized with SAPP or PBS (FIG. 2). In addition, in theselatter three groups there was a characteristic involvement of vulnerablesubregions of the brain classically seen in AD, such as the outermolecular layer of the hippocampal dentate gyrus, in all three of thesegroups.

The brains that contained no Aβ deposits were also devoid of neuriticplaques that are typically visualized in PDAPP mice with the human APPantibody 8E5. All of brains from the remaining groups (SAP-injected, PBSand uninjected mice) had numerous neuritic plaques typical of untreatedPDAPP mice. A small number of neuritic plaques were present in one mousetreated with AN1792, and a single cluster of dystrophic neurites wasfound in a second mouse treated with AN1792. Image analyses of thehippocampus, and shown in FIG. 3, demonstrated the virtual eliminationof dystrophic neurites in AN1792-treated mice (median 0.00%) compared tothe PBS recipients (median 0.28%, p=0.0005).

Astrocytosis characteristic of plaque-associated inflammation was alsoabsent in the brains of the Aβ1-42 injected group. The brains from themice in the other groups contained abundant and clustered GFAP-positiveastrocytes typical of Aβ plaque-associated gliosis. A subset of theGFAP-reacted slides were counter-stained with Thioflavin S to localizethe Aβ deposits. The GFAP-positive astrocytes were associated with Aβplaques in the SAP, PBS and untreated controls. No such association wasfound in the plaque-negative Aβ1-42 treated mice, while minimalplaque-associated gliosis was identified in one mouse treated withAN1792.

Image analyses, shown in FIG. 4 for the retrosplenial cortex, verifiedthat the reduction in astrocytosis was significant with a median valueof 1.56% for those treated with AN1792 versus median values greater than6% for groups immunized with SAP peptides, PBS or untreated (p=0.0017).

Evidence from a subset of the Aβ1-42- and PBS-injected mice indicatedplaque-associated MHC II immunoreactivity was absent in the Aβ1-42injected mice, consistent with lack of an Aβ-related inflammatoryresponse.

Sections of the mouse brains were also reacted with a mAβ specific forMAC-1, a cell surface protein. MAC-1 (CD11b) is an integrin familymember and exists as a heterodimer with CD18. The CD11b/CD18 complex ispresent on monocytes, macrophages, neutrophils and natural killer cells(Mak and Simard). The resident MAC-1-reactive cell type in the brain islikely to be microglia based on similar phenotypic morphology inMAC-1immunoreacted sections. Plaque-associated MAC-1 labeling was lowerin the brains of mice treated with AN1792 compared to the PBS controlgroup, a finding consistent with the lack of an Aβ-induced inflammatoryresponse.

C. Conclusion

The lack of Aβ plaques and reactive neuronal and gliotic changes in thebrains of the Aβ1-42-injected mice indicate that no or extremely littleamyloid was deposited in their brains, and pathological consequences,such as gliosis and neuritic pathology, were absent. PDAPP mice treatedwith Aβ1-42 show essentially the same lack of pathology as controlnontransgenic mice. Therefore, Aβ1-42 injections are highly effective inthe prevention of deposition or clearance of human Aβ from brain tissue,and elimination of subsequent neuronal and inflammatory degenerativechanges. Thus, administration of Aβ peptide has therapeutic benefit inprevention of AD.

EXAMPLE II Dose Response Study

Groups of five-week old, female Swiss Webster mice (N=6 per group) wereimmunized with 300, 100, 33, 11, 3.7, 1.2, 0.4, or 0.13 ug of Aβformulated in CFA/IFA administered intraperitoneally. Three doses weregiven at biweekly intervals followed by a fourth dose one month later.The first dose was emulsified with CFA and the remaining doses wereemulsified with IFA. Animals were bled 4-7 days following eachimmunization starting after the second dose for measurement of antibodytiters. Animals in a subset of three groups, those immunized with 11,33, or 300 μg of antigen, were additionally bled at approximatelymonthly intervals for four months following the fourth immunization tomonitor the decay of the antibody response across a range of vaccinedoses. These animals received a final fifth immunization at seven monthsafter study initiation. They were sacrificed one week later to measureantibody responses to AN1792 and to perform toxicological analyses.

A declining dose response was observed from 300 to 3.7 μg with noresponse at the two lowest doses. Mean antibody titers are about 1:1000after 3 doses and about 1:10,000 after 4 doses of 11-300 μg of antigen(see FIG. 5).

Antibody titers rose dramatically for all but the lowest dose groupfollowing the third immunization with increases in GMTs ranging from 5-to 25-fold. Low antibody responses were then detectable for even the 0.4μg recipients. The 1.2 and 3.7 μg groups had comparable titers with GMTsof about 1000 and the highest four doses clustered together with GMTs ofabout 25,000, with the exception of the 33 μg dose group with a lowerGMT of 3000. Following the fourth immunization, the titer increase wasmore modest for most groups. There was a clear dose response across thelower antigen dose groups from 0.14 μg to 11 μg ranging from nodetectable antibody for recipients of 0.14 μg to a GMT of 36,000 forrecipients of 11 μg. Again, titers for the four highest dose groups of11 to 300 μg clustered together. Thus following two immunizations, theantibody titer was dependent on the antigen dose across the broad rangefrom 0.4 to 300 μg. By the third immunization, titers of the highestfour doses were all comparable and they remained at a plateau after anadditional immunization.

One month following the fourth immunization, titers were 2- to 3-foldhigher in the 300 μg group than those measured from blood drawn fivedays following the immunization (FIG. 6). This observation suggests thatthe peak anamnestic antibody response occurred later than 5 dayspost-immunization. A more modest (50%) increase was seen at this time inthe 33 μg group. In the 300 μg dose group at two months following thelast dose, GMTs declined steeply by about 70%. After another month, thedecline was less steep at 45% (100 μg) and about 14% for the 33 and 11μg doses. Thus, the rate of decline in circulating antibody titersfollowing cessation of immunization appears to be biphasic with a steepdecline the first month following peak response followed by a moremodest rate of decrease thereafter.

The antibody titers and the kinetics of the response of these SwissWebster mice are similar to those of young heterozygous PDAPP transgenicmice immunized in a parallel manner. Dosages effective to induce animmune response in humans are typically similar to dosages effective inmice.

EXAMPLE III Screen For Therapeutic Efficacy Against Established AD

This assay is designed to test immunogenic agents for activity inarresting or reversing neuropathological characteristics of AD in agedanimals. Immunizations with 42 amino acid long Aβ (AN1792) were begun ata timepoint when amyloid plaques are already present in the brains ofthe PDAPP mice.

Over the timecourse used in this study, untreated PDAPP mice develop anumber of neurodegenerative changes that resemble those found in AD(Games et al., supra and Johnson-Wood et al., Proc. Natl. Acad. Sci. USA94, 1550-1555 (1997)). The deposition of Aβ into amyloid plaques isassociated with a degenerative neuronal response consisting of aberrantaxonal and dendritic elements, called dystrophic neurites. Amyloiddeposits that are surrounded by and contain dystrophic neurites calledneuritic plaques. In both AD and the PDAPP mouse, dystrophic neuriteshave a distinctive globular structure, are immunoreactive with a panelof antibodies recognizing APP and cytoskeletal components, and displaycomplex subcellular degenerative changes at the ultrastructural level.These characteristics allow for disease-relevant, selective andreproducible measurements of neuritic plaque formation in the PDAPPbrains. The dystrophic neuronal component of PDAPP neuritic plaques iseasily visualized with an antibody specific for human APP (mAβ 8E5), andis readily measurable by computer-assisted image analysis. Therefore, inaddition to measuring the effects of AN1792 on amyloid plaque formation,we monitored the effects of this treatment on the development ofneuritic dystrophy.

Astrocytes and microglia are non-neuronal cells that respond to andreflect the degree of neuronal injury. GFAP-positive astrocytes and MHCII-positive microglia are commonly observed in AD, and their activationincreases with the severity of the disease. Therefore, we also monitoredthe development of reactive astrocytosis and microgliosis in theAN1792-treated mice.

A. Materials and Methods

Forty-eight, heterozygous female PDAPP mice, 11 to 11.5 months of age,obtained from Charles River, were randomly divided into two groups: 24mice to be immunized with 100 μg of AN1792 and 24 mice to be immunizedwith PBS, each combined with Freund's adjuvant. The AN1792 and PBSgroups were again divided when they reached ˜15 months of age. At 15months of age approximately half of each group of the AN1792- andPBS-treated animals were euthanized (n=10 and 9, respectively), theremainder continued to receive immunizations until termination at ˜18months (n=9 and 12, respectively). A total of 8 animals (5 AN1792, 3PBS) died during the study. In addition to the immunized animals,one-year old (n=10), 15-month old (n=10) and 18-month old (n=10)untreated PDAPP mice were included for comparison in the ELISAs tomeasure Aβ and APP levels in the brain; the one-year old animals werealso included in the immunohistochemical analyses.

Methodology was as in Example 1 unless otherwise indicated. US Peptideslot 12 and California Peptides lot ME0339 of AN1792 were used to preparethe antigen for the six immunizations administered prior to the 15-monthtimepoint. California Peptides lots ME0339 and ME0439 were used for thethree additional immunizations administered between 15 and 18 months.

For immunizations, 100 μg of AN1792 in 200 μl PBS or PBS alone wasemulsified 1:1 (vol:vol) with Complete Freund's adjuvant (CFA) orIncomplete Freund's adjuvant (IFA) or PBS in a final volume of 400 μl.The first immunization was delivered with CFA as adjuvant, the next fourdoses were given with IFA and the final four doses with PBS alonewithout added adjuvant. A total of nine immunizations were given overthe seven-month period on a two-week schedule for the first three dosesfollowed by a four-week interval for the remaining injections. Thefour-month treatment group, euthanized at 15 months of age, receivedonly the first 6 immunizations.

B. Results

1. Effects of AN1792 Treatment on Amyloid Burden

The results of AN1792 treatment on cortical amyloid burden determined byquantitative image analysis are shown in FIG. 7. The median value ofcortical amyloid burden was 0.28% in a group of untreated 12-month oldPDAPP mice, a value representative of the plaque load in mice at thestudy's initiation. At 18 months, the amyloid burden increased over17-fold to 4.87% in PBS-treated mice, while AN1792-treated mice had agreatly reduced amyloid burden of only 0.01%, notably less than the12-month untreated and both the 15- and 18-month PBS-treated groups. Theamyloid burden was significantly reduced in the AN1792 recipients atboth 15 (96% reduction; p=0.003) and 18 (>99% reduction; p=0.0002)months. Typically, cortical amyloid deposition in PDAPP mice initiatesin the frontal and retrosplenial cortices (RSC) and progresses in aventral-lateral direction to involve the temporal and entorhinalcortices (EC). Little or no amyloid was found in the EC of 12 month-oldmice, the approximate age at which AN1792 was first administered. After4 months of AN1792 treatment, amyloid deposition was greatly diminishedin the RSC, and the progressive involvement of the EC was entirelyeliminated by AN1792 treatment. The latter observation showed thatAN1792 completely halted the progression of amyloid that would normallyinvade the temporal and ventral cortices, as well as arrested orpossibly reversed deposition in the RSC.

The profound effects of AN1792 treatment on developing cortical amyloidburden in the PDAPP mice are further demonstrated by the 18-month group,which had been treated for seven months. A near complete absence ofcortical amyloid was found in the AN1792-treated mouse, with a totallack of diffuse plaques, as well as a reduction in compacted deposits.

2. AN1792 Treatment-associated Cellular and Morphological Changes

A population of Aβ-positive cells was found in brain regions thattypically contain amyloid deposits. Remarkably, in several brains fromAN1792 recipients, very few or no extracellular cortical amyloid plaqueswere found. Most of the Aβ immunoreactivity appeared to be containedwithin cells with large lobular or clumped some. Phenotypically, thesecells resembled activated microglia or monocytes. They wereimmunoreactive with antibodies recognizing ligands expressed byactivated monocytes and microglia (MHC II and CD11b) and wereoccasionally associated with the wall or lumen of blood vessels.Comparison of near-adjacent sections labeled with Aβ and MHC II-specificantibodies revealed that similar patterns of these cells were recognizedby both classes of antibodies. Detailed examination of theAN1792-treated brains revealed that the MHC II-positive cells wererestricted to the vicinity of the limited amyloid remaining in theseanimals. Under the fixation conditions employed, the cells were notimmunoreactive with antibodies that recognize T cell (CD3, CD3e) or Bcell (CD45RA, CD45RB) ligands or leukocyte common antigen (CD45), butwere reactive with an antibody recognizing leukosialin (CD43) whichcross-reacts with monocytes. No such cells were found in any of thePBS-treated mice.

PDAPP mice invariably develop heavy amyloid deposition in the outermolecular layer of the hippocampal dentate gyrus. The deposition forms adistinct streak within the perforant pathway, a subregion thatclassically contains amyloid plaques in AD. The characteristicappearance of these deposits in PBS-treated mice resembled thatpreviously characterized in untreated PDAPP mice. The amyloid depositionconsisted of both diffuse and compacted plaques in a continuous band. Incontrast, in a number of brains from AN1792-treated mice this patternwas drastically altered. The hippocampal amyloid deposition no longercontained diffuse amyloid, and the banded pattern was completelydisrupted. Instead, a number of unusual punctate structures were presentthat are reactive with anti-Aβ antibodies, several of which appeared tobe amyloid-containing cells.

MHC II-positive cells were frequently observed in the vicinity ofextracellular amyloid in AN1792-treated animals. The pattern ofassociation of Aβ-positive cells with amyloid was very similar inseveral brains from AN1792-treated mice. The distribution of thesemonocytic cells was restricted to the proximity of the deposited amyloidand was entirely absent from other brain regions devoid of Aβ plaques.

Quantitative image analysis of MHC II and MAC I-labeled sectionsrevealed a trend towards increased immunoreactivity in the RSC andhippocampus of AN1792-treated mice compared to the PBS group whichreached significance with the measure of MAC 1 reactivity inhippocampus.

These results are indicative of active, cell-mediated removal of amyloidin plaque-bearing brain regions.

3. AN1792 Effects on Aβ Levels: ELISA Determinations

(a) Cortical Levels

In untreated PDAPP mice, the median level of total Aβ in the cortex at12 months was 1,600 ng/g, which increased to 8,700 ng/g by 15 months(Table 2). At 18 months the value was 22,000 ng/g, an increase of over10-fold during the time course of the experiment. PBS-treated animalshad 8,600 ng/g total Aβ at 15 months which increased to 19,000 ng/g at18 months. In contrast, AN1792-treated animals had 81% less total Aβ at15 months (1,600 ng/g) than the PBS-immunized group. Significantly less(p=0.0001) total Aβ (5,200 ng/g) was found at 18 months when the AN1792and PBS groups were compared (Table 2), representing a 72% reduction inthe Aβ that would otherwise be present. Similar results were obtainedwhen cortical levels of Aβ42 were compared, namely that theAN1792-treated group contained much less Aβ42, but in this case thedifferences between the AN1792 and PBS groups were significant at both15 months (p=0.04) and 18 months (p=0.0001, Table 2).

TABLE 2 Median Aβ Levels (ng/g) in Cortex UNTREATED PBS AN1792 TotalTotal Total Age Aβ Aβ42 (n) Aβ Aβ42 (n) Aβ Aβ42 (n) 12  1,600  1,300(10) 15  8,700  8,300 (10)  8,600  7,200  (9) 1,600  1,300*  (10) 1822,200 18,500 (10) 19,000 15,900 (12) 5,200** 4,000**  (9) *p = 0.0412**p = 0.0001(b) Hippocampal Levels

In untreated PDAPP mice, median hippocampal levels of total Aβ at twelvemonths of age were 15,000 ng/g which increased to 51,000 ng/g at 15months and further to 81,000 ng/g at 18 months (Table 3). Similarly, PBSimmunized mice showed values of 40,000 ng/g and 65,000 ng/g at 15 monthsand 18 months, respectively. AN1792 immunized animals exhibited lesstotal Aβ, specifically 25,000 ng/g and 51,000 ng/g at the respective15-month and 18-month timepoints. The 18-month AN1792-treated groupvalue was significantly lower than that of the PBS treated group(p=0.0105; Table 3). Measurement of Aβ42 gave the same pattern ofresults, namely that levels in the AN1792-treated group weresignificantly lower than in the PBS group (39,000 ng/g vs. 57,000 ng/g,respectively; p=0.0022) at the 18-month evaluation (Table 3).

TABLE 3 Median Aβ Levels (ng/g) in Hippocampus UNTREATED PBS AN1792Total Total Total Age Aβ Aβ42 (n) Aβ Aβ42 (n) Aβ Aβ42 (n) 12 15,50011,100 (10) 15 51,500 44,400 (10) 40,100 35,700  (9) 24,500  22,100 (10) 18 80,800 64,200 (10) 65,400 57,100 (12) 50,900* 38,900**  (9) *p =0.0105 **p = 0.0022(c) Cerebellar Levels

In 12-month untreated PDAPP mice, the median cerebellar level of totalAβ was 15 ng/g (Table 4). At 15 months, this median increased to 28 ng/gand by 18 months had risen to 35 ng/g. PBS-treated animals displayedmedian total Aβ values of 21 ng/g at 15 months and 43 ng/g at 18 months.AN1792-treated animals were found to have 22 ng/g total Aβ at 15 monthsand significantly less (p=0.002) total Aβ at 18 months (25 ng/g) thanthe corresponding PBS group (Table 4).

TABLE 4 Median Aβ Levels (ng/g) in Cerebellum UNTREATED PBS AN1792 AgeTotal Aβ (n) Total Aβ (n) Total Aβ (n) 12 15.6 (10) 15 27.7 (10) 20.8 (9) 21.7  (10) 15 35.0 (10) 43.1 (12) 24.8*  (9) *p = 0.0018

4. Effects of AN1792 Treatment on APP Levels

APP-α and the full-length APP molecule both contain all or part of theAβ sequence and thus could be potentially impacted by the generation ofan AN1792-directed immune response. In studies to date, a slightincrease in APP levels has been noted as neuropathology increases in thePDAPP mouse. In the cortex, levels of either APP-α/FL (full length) orAPP-α were essentially unchanged by treatment with the exception thatAPP-α was reduced by 19% at the 18-month timepoint in the AN1792-treatedvs. the PBS-treated group. The 18-month AN1792-treated APP values werenot significantly different from values of the 12-month and 15-monthuntreated and 15-month PBS groups. In all cases the APP values remainedwithin the ranges that are normally found in PDAPP mice.

5. Effects of AN1792 Treatment on Neurodegenerative and GlioticPathology

Neuritic plaque burden was significantly reduced in the frontal cortexof AN1792-treated mice compared to the PBS group at both 15 (84%;p=0.03) and 18 (55%; p=0.01) months of age (FIG. 8). The median value ofthe neuritic plaque burden increased from 0.32% to 0.49% in the PBSgroup between 15 and 18 months of age. This contrasted with the greatlyreduced development of neuritic plaques in the AN1792 group, with medianneuritic plaque burden values of 0.05% and 0.22%, in the 15 and 18 monthgroups, respectively.

Immunizations with AN1792 seemed well tolerated and reactiveastrocytosis was also significantly reduced in the RSC of AN1792-treatedmice when compared to the PBS group at both 15 (56%; p=0.011) and 18(39%; p=0.028) months of age (FIG. 9). Median values of the percent ofastrocytosis in the PBS group increased between 15 and 18 months from4.26% to 5.21%. AN1792-treatment suppressed the development ofastrocytosis at both time points to 1.89% and 3.2%, respectively. Thissuggests the neuropil was not being damaged by the clearance process.

6. Antibody Responses

As described above, eleven-month old, heterozygous PDAPP mice (N=24)received a series of 5 immunizations of 100 μg of AN1792 emulsified withFreund's adjuvant and administered intraperitoneally at weeks 0, 2, 4,8, and 12, and a sixth immunization with PBS alone (no Freund'sadjuvant) at week 16. As a negative control, a parallel set of 24age-matched transgenic mice received immunizations of PBS emulsifiedwith the same adjuvants and delivered on the same schedule. Animals werebled within three to seven days following each immunization startingafter the second dose. Antibody responses to AN1792 were measured byELISA. Geometric mean titers (GMT) for the animals that were immunizedwith AN1792 were approximately 1,900, 7,600, and 45,000 following thesecond, third and last (sixth) doses respectively. No Aβ-specificantibody was measured in control animals following the sixthimmunization.

Approximately one-half of the animals were treated for an additionalthree months, receiving immunizations at about 20, 24 and 27 weeks. Eachof these doses was delivered in PBS vehicle alone without Freund'sadjuvant. Mean antibody titers remained unchanged over this time period.In fact, antibody titers appeared to remain stable from the fourth tothe eighth bleed corresponding to a period covering the fifth to theninth injections.

To determine if the Aβ-specific antibodies elicited by immunization thatwere detected in the sera of AN1792-treated mice were also associatedwith deposited brain amyloid, a subset of sections from the AN1792- andPBS-treated mice were reacted with an antibody specific for mouse IgG.In contrast to the PBS group, Aβ plaques in AN1792-treated brains werecoated with endogenous IgG. This difference between the two groups wasseen in both 15- and 18-month groups. Particularly striking was the lackof labeling in the PBS group, despite the presence of a heavy amyloidburden in these mice. These results show that immunization with asynthetic Aβ protein generates antibodies that recognize and bind invivo to the Aβ in amyloid plaques.

7. Cellular-mediated Immune Responses

Spleens were removed from nine AN1792-immunized and 12 PBS-immunized18-month old PDAPP mice 7 days after the ninth immunization. Splenocyteswere isolated and cultured for 72 h in the presence of Aβ40, Aβ42, orAβ40-1 (reverse order protein). The mitogen Con A served as a positivecontrol. Optimum responses were obtained with >1.7 μM protein. Cellsfrom all nine AN1792-treated animals proliferated in response to eitherA≢1-40 or Aβ1-42 protein, with equal levels of incorporation for bothproteins (FIG. 10A). There was no response to the Aβ40-1 reverseprotein. Cells from control animals did not respond to any of the Aβproteins (FIG. 10B).

Conclusion

The results of this study show that AN1792 immunization of PDAPP micepossessing existing amyloid deposits slows and prevents progressiveamyloid deposition and retard consequential neuropathological changes inthe aged PDAPP mouse brain. Immunizations with AN1792 essentially haltedamyloid developing in structures that would normally succumb toamyloidosis. Thus, administration of Aβ peptide has therapeutic benefitin the treatment of Aβ.

EXAMPLE IV Screen of Aβ Fragments

100 PDAPP mice age 7-9 months are immunized with 9 different regions ofAPP and Aβ to determine which epitopes convey the response. The 9different immunogens and one control are injected i.p. as describedabove. The immunogens include four human Aβ peptide conjugates 1-12,13-28, 32-42, 1-5, all coupled to sheep anti-mouse IgG via a cystinelink; an APP polypeptide aa 592-695, aggregated human Aβ1-40, andaggregated human Aβ 25-35, and aggregated rodent Aβ42. Aggregated Aβ42and PBS are used as controls. Ten mice are used per treatment group.Titers are monitored as above and mice are euthanized at the end of 4months of injections. Histochemistry, Aβ levels, and toxicology aredetermined post mortem.

A. Materials and Methods

1. Preparation of Immunogens

Preparation of coupled Aβ peptides: four human Aβ peptide conjugates(amino acid residues 1-5, 1-12, 13-28, and 33-42, each conjugated tosheep anti-mouse IgG) were prepared by coupling through an artificialcysteine added to the Aβ peptide using the crosslinking reagentsulfo-EMCS. The Aβ peptide derivatives were synthesized with thefollowing final amino acid sequences. In each case, the location of theinserted cysteine residue is indicated by underlining. The Aβ13-28peptide derivative also had two glycine residues added prior to thecarboxyl terminal cysteine as indicated.

-   -   Aβ1-12 peptide NH₂-DAEFRHDSGYEVC-COOH (SEQ ID NO:2)    -   Aβ1-5 peptide NH₂-DAEFRC-COOH (SEQ ID) NO:3)    -   Aβ33-42 peptide NH₂-C-amino-heptanoic acid-GLMVGGVVIA-COOH (SEQ        ID NO:4)    -   Aβ13-28 peptide Ac-NH-HHQKLVFFAEDVGSNKGGC-COOH (SEQ ID NO:5)

To prepare for the coupling reaction, ten mg of sheep anti-mouse IgG(Jackson ImmunoResearch Laboratories) was dialyzed overnight against 10mM sodium borate buffer, pH 8.5. The dialyzed antibody was thenconcentrated to a volume of 2 mL using an Amicon Centriprep tube. Ten mgsulfo-EMCS.

[N (ε-maleimidocuproyloxy)succinimide] (Molecular Sciences Co.) wasdissolved in one mL deionized water. A 40-fold molar excess ofsulfo-EMCS was added dropwise with stirring to the sheep anti-mouse IgGand then the solution was stirred for an additional ten min. Theactivated sheep anti-mouse IgG was purified and buffer exchanged bypassage over a 10 mL gel filtration column (Pierce Presto Column,obtained from Pierce Chemicals) equilibrated with 0.1 M NaPO₄, 5 mMEDTA, pH 6.5. Antibody containing fractions, identified by absorbance at280 nm, were pooled and diluted to a concentration of approximately 1mg/mL, using 1.4 mg per OD as the extinction coefficient. A 40-foldmolar excess of Aβ peptide was dissolved in 20 mL of 10 mM NaPO₄, pH8.0, with the exception of the Aβ33-42 peptide for which 10 mg was firstdissolved in 0.5 mL of DMSO and then diluted to 20 mL with the 10 mMNaPO₄ buffer. The peptide solutions were each added to 10 mL ofactivated sheep anti-mouse IgG and rocked at room temperature for 4 hr.The resulting conjugates were concentrated to a final volume of lessthan 10 mL using an Amicon Centriprep tube and then dialyzed against PBSto buffer exchange the buffer and remove free peptide. The conjugateswere passed through 0.22 μ-pore size filters for sterilization and thenaliquoted into fractions of 1 mg and stored frozen at −20° C. Theconcentrations of the conjugates were determined using the BCA proteinassay (Pierce Chemicals) with horse IgG for the standard curve.Conjugation was documented by the molecular weight increase of theconjugated peptides relative to that of the activated sheep anti-mouseIgG. The Aβ1-5 sheep anti-mouse conjugate was a pool of twoconjugations, the rest were from a single preparation.

2. Preparation of Aggregated Aβ Peptides

Human 1-40 (AN1528; California Peptides Inc., Lot ME0541), human 1-42(AN1792; California Peptides Inc., Lots ME0339 and ME0439), human 25-35,and rodent 1-42 (California Peptides Inc., Lot ME0218) peptides werefreshly solubilized for the preparation of each set of injections fromlyophilized powders that had been stored desiccated at −20° C. For thispurpose, two mg of peptide were added to 0.9 ml of deionized water andthe mixture was vortexed to generate a relatively uniform solution orsuspension. Of the four, AN1528 was the only peptide soluble at thisstep. A 100 μl aliquot of 10×PBS (1×PBS: 0.15 M NaCl, 0.01 M sodiumphosphate, pH 7.5) was then added at which point AN1528 began toprecipitate. The suspension was vortexed again and incubated overnightat 37° C. for use the next day.

Preparation of the pBx6 protein: An expression plasmid encoding pBx6, afusion protein consisting of the 100-amino acid bacteriophage MS-2polymerase N-terminal leader sequence followed by amino acids 592-695 ofAPP (βAPP) was constructed as described by Oltersdorf et al., J. Biol.Chem. 265, 4492-4497 (1990). The plasmid was transfected into E. coliand the protein was expressed after induction of the promoter. Thebacteria were lysed in 8M urea and pBx6 was partially purified bypreparative SDS PAGE. Fractions containing pBx6 were identified byWestern blot using a rabbit anti-pBx6 polyclonal antibody, pooled,concentrated using an Amicon Centriprep tube and dialysed against PBS.The purity of the preparation, estimated by Comassie Blue stained SDSPAGE, was approximately 5 to 10%.

B. Results and Discussion 1. Study Design

One hundred male and female, nine- to eleven-month old heterozygousPDAPP transgenic mice were obtained from Charles River Laboratory andTaconic Laboratory. The mice were sorted into ten groups to be immunizedwith different regions of Aβ or APP combined with Freund's adjuvant.Animals were distributed to match the gender, age, parentage and sourceof the animals within the groups as closely as possible. The immunogensincluded four Aβ peptides derived from the human sequence, 1-5, 1-12,13-28, and 33-42, each conjugated to sheep anti-mouse IgG; fouraggregated Aβ peptides, human 1-40 (AN1528), human 1-42 (AN1792), human25-35, and rodent 1-42; and a fusion polypeptide, designated as pBx6,containing APP amino acid residues 592-695. A tenth group was immunizedwith PBS combined with adjuvant as a control.

For each immunization, 100 μg of each Aβ peptide in 200 μl PBS or 200 μgof the APP derivative pBx6 in the same volume of PBS or PBS alone wasemulsified 1:1 (vol:vol) with Complete Freund's adjuvant (CFA) in afinal volume of 400 μl for the first immunization, followed by a boostof the same amount of immunogen in Incomplete Freund's adjuvant (IFA)for the subsequent four doses and with PBS for the final dose.Immunizations were delivered intraperitoneally on a biweekly schedulefor the first three doses, then on a monthly schedule thereafter.Animals were bled four to seven days following each immunizationstarting after the second dose for the measurement of antibody titers.Animals were euthanized approximately one week after the final dose.

2. Aβ and APP Levels in the Brain

Following about four months of immunization with the various Aβ peptidesor the APP derivative, brains were removed from saline-perfused animals.One hemisphere was prepared for immunohistochemical analysis and thesecond was used for the quantitation of Aβ and APP levels. To measurethe concentrations of various forms of beta amyloid peptide and amyloidprecursor protein, the hemisphere was dissected and homogenates of thehippocampal, cortical, and cerebellar regions were prepared in 5 Mguanidine. These were diluted and the level of amyloid or APP wasquantitated by comparison to a series of dilutions of standards of Aβpeptide or APP of known concentrations in an ELISA format.

The median concentration of total Aβ for the control group immunizedwith PBS was 5.8-fold higher in the hippocampus than in the cortex(median of 24,318 ng/g hippocampal tissue compared to 4,221 ng/g for thecortex). The median level in the cerebellum of the control group (23.4ng/g tissue) was about 1,000-fold lower than in the hippocampus. Theselevels are similar to those that we have previously reported forheterozygous PDAPP transgenic mice of this age (Johnson-Woods et al.,1997, supra).

For the cortex, a subset of treatment groups had median total Aβ andAβ1-42 levels which differed significantly from those of the controlgroup (p<0.05), those animals receiving AN1792, rodent Aβ1-42 or theAβ1-5 peptide conjugate as shown in FIG. 11. The median levels of totalAβ were reduced by 75%, 79% and 61%, respectively, compared to thecontrol for these treatment groups. There were no discernablecorrelations between Aβ-specific antibody titers and Aβ levels in thecortical region of the brain for any of the groups.

In the hippocampus, the median reduction of total Aβ associated withAN1792 treatment (46%, p=0.0543) was not as great as that observed inthe cortex (75%, p=0.0021). However, the magnitude of the reduction wasfar greater in the hippocampus than in the cortex, a net reduction of11,186 ng/g tissue in the hippocampus versus 3,171 ng/g tissue in thecortex. For groups of animals receiving rodent Aβ1-42 or Aβ1-5, themedian total Aβ levels were reduced by 36% and 26%, respectively.However, given the small group sizes and the high variability of theamyloid peptide levels from animal to animal within both groups, thesereductions were not significant. When the levels of Aβ1-42 were measuredin the hippocampus, none of the treatment-induced reductions reachedsignificance. Thus, due to the smaller Aβ burden in the cortex, changesin this region are a more sensitive indicator of treatment effects. Thechanges in Aβ levels measured by ELISA in the cortex are similar, butnot identical, to the results from the immunohistochemical analysis (seebelow).

Total Aβ was also measured in the cerebellum, a region typicallyunaffected in the AD pathology. None of the median Aβ concentrations ofany of the groups immunized with the various Aβ peptides or the APPderivative differed from that of the control group in this region of thebrain. This result suggests that non-pathological levels of Aβ areunaffected by treatment.

APP concentration was also determined by ELISA in the cortex andcerebellum from treated and control mice. Two different APP assays wereutilized. The first, designated APP-α/FL, recognizes both APP-alpha (α,the secreted form of APP which has been cleaved within the Aβ sequence),and full-length forms (FL) of APP, while the second recognizes onlyAPP-α. In contrast to the treatment-associated diminution of Aβ in asubset of treatment groups, the levels of APP were unchanged in all ofthe treated compared to the control animals. These results indicate thatthe immunizations with Aβ peptides are not depleting APP; rather thetreatment effect is specific to Aβ.

In summary, total Aβ and Aβ1-42 levels were significantly reduced in thecortex by treatment with AN1792, rodent Aβ1-42 or Aβ1-5 conjugate. Inthe hippocampus, total Aβ was significantly reduced only by AN1792treatment. No other treatment-associated changes in Aβ or APP levels inthe hippocampal, cortical or cerebellar regions were significant.

3. Histochemical Analyses

Brains from a subset of six groups were prepared for immunohistochemicalanalysis, three groups immunized with the Aβ peptide conjugates Aβ1-5,Aβ1-12, and Aβ13-28; two groups immunized with the full length Aβaggregates AN1792 and AN1528 and the PBS-treated control group. Theresults of image analyses of the amyloid burden in brain sections fromthese groups are shown in FIG. 12. There were significant reductions ofamyloid burden in the cortical regions of three of the treatment groupsversus control animals. The greatest reduction of amyloid burden wasobserved in the group receiving AN1792 where the mean value was reducedby 97% (p=0.001). Significant reductions were also observed for thoseanimals treated with AN1528 (95%, p=0.005) and the Aβ1-5 peptideconjugate (67%, p=0.02).

The results obtained by quantitation of total Aβ or Aβ1-42 by ELISA andamyloid burden by image analysis differ to some extent. Treatment withAN1528 had a significant impact on the level of cortical amyloid burdenwhen measured by quantitative image analysis but not on theconcentration of total Aβ in the same region when measured by ELISA. Thedifference between these two results is likely to be due to thespecificities of the assays. Image analysis measures only insoluble Aβaggregated into plaques. In contrast, the ELISA measures all forms ofAβ, both soluble and insoluble, monomeric and aggregated. Since thedisease pathology is thought to be associated with the insolubleplaque-associated form of Aβ, the image analysis technique may have moresensitivity to reveal treatment effects. However since the ELISA is amore rapid and easier assay, it is very useful for screening purposes.Moreover it may reveal that the treatment-associated reduction of Aβ isgreater for plaque-associated than total Aβ.

To determine if the Aβ-specific antibodies elicited by immunization inthe treated animals reacted with deposited brain amyloid, a subset ofthe sections from the treated animals and the control mice were reactedwith an antibody specific for mouse IgG. In contrast to the PBS group,Aβ-containing plaques were coated with endogenous IgG for animalsimmunized with the Aβ peptide conjugates Aβ1-5, Aβ1-12, and Aβ13-28; andthe full length Aβ aggregates AN1792 and AN1528. Brains from animalsimmunized with the other Aβ peptides or the APP peptide pBx6 were notanalyzed by this assay.

4. Measurement of Antibody Titers

Mice were bled four to seven days following each immunization startingafter the second immunization, for a total of five bleeds. Antibodytiters were measured as Aβ1-42-binding antibody using a sandwich ELISAwith plastic multi-well plates coated with Aβ1-42. As shown in FIG. 13,peak antibody titers were elicited following the fourth dose for thosefour vaccines which elicited the highest titers of AN1792-specificantibodies: AN1792 (peak GMT: 94,647), AN1528 (peak GMT: 88,231), Aβ1-12conjugate (peak GMT: 47,216)and rodent Aβ1-42 (peak GMT: 10,766). Titersfor these groups declined somewhat following the fifth and sixth doses.For the remaining five immunogens, peak titers were reached followingthe fifth or the sixth dose and these were of much lower magnitude thanthose of the four highest titer groups: Aβ1-5 conjugate (peak GMT:2,356), pBx6 (peak GMT: 1,986), Aβ13-28 conjugate (peak GMT: 1,183),Aβ33-42 conjugate (peak GMT: 658), Aβ25-35 (peak GMT: 125). Antibodytiters were also measured against the homologous peptides using the sameELISA sandwich format for a subset of the immunogens, those groupsimmunized with Aβ1-5, Aβ13-28, Aβ25-35, Aβ33-42 or rodent Aβ1-42. Thesetiters were about the same as those measured against Aβ1-42 except forthe rodent Aβ1-42 immunogen in which case antibody titers against thehomologous immunogen were about two-fold higher. The magnitude of theAN1792-specific antibody titer of individual animals or the mean valuesof treatment groups did not correlate with efficacy measured as thereduction of Aβ in the cortex.

5. Lymphoproliferative Responses

Aβ-dependent lymphoproliferation was measured using spleen cellsharvested approximately one week following the final, sixth,immunization. Freshly harvested cells, 10⁵ per well, were cultured for 5days in the presence of Aβ1-40 at a concentration of 5 μM forstimulation. Cells from a subset of seven of the ten groups were alsocultured in the presence of the reverse peptide, Aβ40-1. As a positivecontrol, additional cells were cultured with the T cell mitogen, PHA,and, as a negative control, cells were cultured without added peptide.

Lymphocytes from a majority of the animals proliferated in response toPHA. There were no significant responses to the Aβ40-1 reverse peptide.Cells from animals immunized with the larger aggregated Aβ peptides,AN1792, rodent Aβ1-42 and AN1528 proliferated robustly when stimulatedwith Aβ1-40 with the highest cpm in the recipients of AN1792. One animalin each of the groups immunized with Aβ1-12 conjugate, Aβ13-28 conjugateand Aβ25-35 proliferated in response to Aβ1-40. The remaining groupsreceiving Aβ1-5 conjugate, Aβ33-42 conjugate pBx6 or PBS had no animalswith an Aβ-stimulated response. These results are summarized in Table 5below.

TABLE 5 Immunogen Conjugate Aβ Amino Acids Responders Aβ1-5 yes  5-mer0/7 Aβ1-12 yes 12-mer 1/8 Aβ13-28 yes 16-mer 1/9 Aβ25-35 11-mer 1/9Aβ33-42 yes 10-mer  0/10 Aβ1-40 40-mer 5/8 Aβ1-42 42-mer 9/9 r Aβ1-4242-mer 8/8 pBx6 0/8 PBS  0-mer 0/8

These results show that AN1792 and AN1528 stimulate strong T cellresponses, most likely of the CD4⁺ phenotype. The absence of anAβ-specific T cell response in animals immunized with Aβ1-5 is notsurprising since peptide epitopes recognized by CD4⁺ T cells are usuallyabout 15 amino acids in length, although shorter peptides can sometimesfunction with less efficiency. Thus the majority of helper T cellepitopes for the four conjugate peptides are likely to reside in the IgGconjugate partner, not in the Aβ region. This hypothesis is supported bythe very low incidence of proliferative responses for animals in each ofthese treatment groups. Since the Aβ1-5 conjugate was effective atsignificantly reducing the level of Aβ in the brain, in the apparentabsence of Aβ-specific T cells, the key effector immune response inducedby immunization with this peptide appears to be antibody.

Lack of T-cell and low antibody response from fusion peptide pBx6,encompassing APP amino acids 592-695 including all of the Aβ residuesmay be due to the poor immunogenicity of this particular preparation.The poor immunogenicity of the Aβ25-35 aggregate is likely due to thepeptide being too small to be likely to contain a good T cell epitope tohelp the induction of an antibody response. If this peptide wereconjugated to a carrier protein, it would probably be more immunogenic.

EXAMPLE V Preparation of Polyclonal Antibodies for Passive Protection

20 non-transgenic mice are immunized with Aβ or other immunogen,optionally plus adjuvant, and are euthanized at 4-5 months. Blood iscollected from immunized mice. Optionally, IgG is separated from otherblood components. Antibody specific for the immunogen may be partiallypurified by affinity chromatography. An average of about 0.5-1 mg ofimmunogen-specific antibody is obtained per mouse, giving a total of5-10 mg.

EXAMPLE VI Passive Immunization with Antibodies to Aβ

Groups of 7-9 month old PDAPP mice each are injected with 0.5 mg in PBSof polyclonal anti-Aβ or specific anti-Aβ monoclonals as shown below.The cell line designated RB44-10D5.19.21 producing the antibody 10D5 hasthe ATCC accession number PTA-5129, having been deposited on Apr. 8,2003. All antibody preparations are purified to have low endotoxinlevels. Monoclonals can be prepared against a fragment by injecting thefragment or longer form of Aβ into a mouse, preparing hybridomas andscreening the hybridomas for an antibody that specifically binds to adesired fragment of Aβ without binding to other nonoverlapping fragmentsof Aβ.

TABLE 6 Antibody Epitope 2H3 Aβ  1-12 10D5 Aβ  1-12 266 Aβ 13-28 21F12Aβ 33-42 Mouse polyclonal Anti-Aggregated Aβ42 anti-human AB42

Mice are injected ip as needed over a 4 month period to maintain acirculating antibody concentration measured by ELISA titer of greaterthan 1/1000 defined by ELISA to Aβ42 or other immunogen. Titers aremonitored as above and mice are euthanized at the end of 4 months ofinjections. Histochemistry, Aβ levels and toxicology are performed postmortem. Ten mice are used per group.

EXAMPLE VII Comparison of Different Adjuvants

This examples compares CFA, alum, an oil-in water emulsion and MPL forcapacity to stimulate an immune response.

A. Materials and Methods

1. Study Design

One hundred female Hartley strain six-week old guinea pigs, obtainedfrom Elm Hill, were sorted into ten groups to be immunized with AN1792or a palmitoylated derivative thereof combined with various adjuvants.Seven groups received injections of AN1792 (33 μg unless otherwisespecified) combined with a) PBS, b) Freund's adjuvant, c) MPL, d)squalene, e) MPL/squalene f) low dose alum, or g) high dose alum (300 μgAN1792). Two groups received injections of a palmitoylated derivative ofAN1792 (33 μg) combined with a) PBS or b) squalene. A final, tenth groupreceived PBS alone without antigen or additional adjuvant. For the groupreceiving Freund's adjuvant, the first dose was emulsified with CFA andthe remaining four doses with IFA. Antigen was administered at a dose of33 μg for all groups except the high dose alum group, which received 300μg of AN1792. Injections were administered intraperitoneally for CFA/IFAand intramuscularly in the hind limb quadriceps alternately on the rightand left side for all other groups. The first three doses were given ona biweekly schedule followed by two doses at a monthly interval). Bloodwas drawn six to seven days following each immunization, starting afterthe second dose, for measurement of antibody titers.

2. Preparation of Immunogens

Two mg Aβ42 (California Peptide, Lot ME0339) was added to 0.9 ml ofdeionized water and the mixture was vortexed to generate a relativelyuniform suspension. A 100 μl aliquot of 10×PBS (1×PBS, 0.15 M NaCl, 0.01M sodium phosphate, pH 7.5) was added. The suspension was vortexed againand incubated overnight at 37° C. for use the next day. Unused AD1-42was stored with desiccant as a lyophilized powder at −20° C.

To the amino terminal residue of AN1792 prior to removal of the nascentpeptide from the resin by treatment with A palmitoylated derivative ofAN1792 was prepared by coupling palmitic anhydride, dissolved indimethyl formamide, hydrofluoric acid.

To prepare vaccine doses with Complete Freund's adjuvant (CFA) (group2), 33 μg of AN1792 in 200 μl PBS was emulsified 1:1 (vol:vol) with CFAin a final volume of 400 μl for the first immunization. For subsequentimmunizations, the antigen was similarly emulsified with IncompleteFreund's adjuvant (IFA).

To prepare vaccine doses with MPL for groups 5 and 8, lyophilized powder(Ribi ImmunoChem Research, Inc., Hamilton, Mont.) was added to 0.2%aqueous triethylamine to a final concentration of 1 mg/ml and vortexed.The mixture was heated to 65 to 70° C. for 30 sec to create a slightlyopaque uniform suspension of micelles. The solution was freshly preparedfor each set of injections. For each injection in group 5, 33 μg ofAN1792 in 16.5 μl PBS, 50 μg of MPL (50 μl) and 162 μl of PBS were mixedin a borosilicate tube immediately before use.

To prepare vaccine doses with the low oil-in-water emulsion, AN1792 inPBS was added to 5% squalene, 0.5% Tween 80, 0.5% Span 85 in PBS toreach a final single dose concentration of 33 μg AN1792 in 250 μl (group6). The mixture was emulsified by passing through a two-chamberedhand-held device 15 to 20 times until the emulsion droplets appeared tobe about equal in diameter to a 1.0 μm diameter standard latex bead whenviewed under a microscope. The resulting suspension was opalescent,milky white. The emulsions were freshly prepared for each series ofinjections. For group 8, MPL in 0.2% triethylamine was added at aconcentration of 50 μg per dose to the squalene and detergent mixturefor emulsification as noted above. For the palmitoyl derivative (group7), 33 μg per dose of palmitoyl-NH-Aβ1-42 was added to squalene andvortexed. Tween 80 and Span 85 were then added with vortexing. Thismixture was added to PBS to reach final concentrations of 5% squalene,0.5% Tween 80, 0.5% Span 85 and the mixture was emulsified as notedabove.

To prepare vaccine doses with alum (groups 9 and 10), AN1792 in PBS wasadded to Alhydrogel (aluminum hydroxide gel, Accurate, Westbury, N.Y.)to reach concentrations of 33 μg (low dose, group 9) or 300 μg (highdose, group 10) AN1792 per 5 mg of alum in a final dose volume of 250μl. The suspension was gently mixed for 4 hr at RT.

3. Measurement of Antibody Titers

Guinea pigs were bled six to seven days following immunization startingafter the second immunization for a total of four bleeds. Antibodytiters against Aβ42 were measured by ELISA as described in GeneralMaterials and Methods.

4. Tissue Preparation

After about 14 weeks, all guinea pigs were administered CO₂.Cerebrospinal fluid was collected and the brains were removed and threebrain regions (hippocampus, cortex and cerebellum) were dissected andused to measure the concentration of total Aβ protein using ELISA.

B. Results

1. Antibody Responses

There was a wide range in the potency of the various adjuvants whenmeasured as the antibody response to AN1792 following immunization. Asshown in FIG. 14, when AN1792 was administered in PBS, no antibody wasdetected following two or three immunizations and negligible responseswere detected following the fourth and fifth doses with geometric meantiters (GMTs) of only about 45. The o/w emulsion induced modest titersfollowing the third dose (GMT 255) that were maintained following thefourth dose (GMT 301) and fell with the final dose (GMT 54). There was aclear antigen dose response for AN1792 bound to alum with 300 μg beingmore immunogenic at all time points than 33 μg. At the peak of theantibody response, following the fourth immunization, the differencebetween the two doses was 43% with GMTs of about 1940 (33 μg) and 3400(300 μg). The antibody response to 33 μg AN1792 plus MPL was verysimilar to that generated with almost a ten-fold higher dose of antigen(300 μg) bound to alum. The addition of MPL to an o/w emulsion decreasedthe potency of the vaccine relative to that with MPL as the soleadjuvant by as much as 75%. A palmitoylated derivative of AN1792 wascompletely non-immunogenic when administered in PBS and gave modesttiters when presented in an o/w emulsion with GMTs of 340 and 105 forthe third and fourth bleeds. The highest antibody titers were generatedwith Freund's adjuvant with a peak GMT of about 87,000, a value almost30-fold greater than the GMTs of the next two most potent vaccines, MPLand high dose AN1792/alum.

The most promising adjuvants identified in this study are MPL and alum.Of these two, MPL appears preferable because a 10-fold lower antigendose was required to generate the same antibody response as obtainedwith alum. The response can be increased by increasing the dose ofantigen and /or adjuvant and by optimizing the immunization schedule.The o/w emulsion was a very weak adjuvant for AN1792 and adding an o/wemulsion to MPL adjuvant diminished the intrinsic adjuvant activity ofMPL alone.

2. Aβ Levels in the Brain

At about 14 weeks the guinea pigs were deeply anesthetized, thecerebrospinal fluid (CSF) was drawn and brains were excised from animalsin a subset of the groups, those immunized with Freund's adjuvant (group2), MPL (group 5), alum with a high dose, 300 μg, of AN1792 (group 10)and the PBS immunized control group (group 3). To measure the level ofAβ peptide, one hemisphere was dissected and homogenates of thehippocampal, cortical, and cerebellar regions were prepared in 5 Mguanidine. These were diluted and quantitated by comparison to a seriesof dilutions of Aβ standard protein of known concentrations in an ELISAformat. The levels of Aβ protein in the hippocampus, the cortex and thecerebellum were very similar for all four groups despite the wide rangeof antibody responses to Aβ elicited by these vaccines. Mean Aβ levelsof about 25 ng/g tissue were measured in the hippocampus, 21 ng/g in thecortex, and 12 ng/g in the cerebellum. Thus, the presence of a highcirculating antibody titer to Aβ for almost three months in some ofthese animals did not alter the total Aβ levels in their brains. Thelevels of Aβ in the CSF were also quite similar between the groups. Thelack of large effect of AN1792 immunization on endogenous Aβ indicatesthat the immune response is focused on pathological formations of Aβ.

EXAMPLE VII Response to Different Adjuvants in Mice

Six-week old female Swiss Webster mice were used for this study with10-13 animals per group. Immunizations were given on days 0, 14, 28, 60,90 and 20 administered subcutaneously in a dose volume of 200 μl. PBSwas used as the buffer for all formulations. Animals were bleed sevendays following each immunization starting after the second dose foranalysis of antibody titers by ELISA. The treatment regime of each groupis summarized in Table 7.

TABLE 7 Experimental Design of Betabloc Study 010 Group N^(a)Adjuvant^(b) Dose Antigen Dose (μg)  1 10 MPL 12.5 μg AN1792 33  2 10MPL 25 μg AN1792 33  3 10 MPL 50 μg AN1792 33  4 13 MPL 125 μg AN1792 33 5 13 MPL 50 μg AN1792 150   6 13 MPL 50 μg AN1528 33  7 10 PBS AN179233  8 10 PBS none  9 10 Squalene 5% AN1792 33 emulsified 10 10 Squalene5% AN1792 33 admixed 11 10 Alum 2 mg AN1792 33 12 13 MPL + Alum 50 μg/AN1792 33 2 mg 13 10 QS21 5 μg AN1792 33 14 10 QS21 10 μg AN1792 33 1510 QS21 25 μg AN1792 33 16 13 QS21 25 μg AN1792 150  17 13 QS21 25 μgAN1528 33 18 13 QS21 + MPL 25 μg/ AN1792 33 50 μg 19 13 QS21 + Alum 25μg/ AN1792 33 2 mg Footnotes: ^(a)Number of mice in each group at theinitiation of the experiment. ^(b)The adjuvants are noted. The bufferfor all these formulations was PBS. For group 8, there was no adjuvantand no antigen.

The ELISA titers of antibodies against Aβ42 in each group are shown inTable 8 below.

TABLE 8 Geometric Mean Antibody Titers Week of Bleed Treatment Group 2.95.0 8.7 12.9 16.7  1 248 1797 2577 6180 4177  2 598 3114 3984 5287 6878 3 1372 5000 7159 12333 12781  4 1278 20791 14368 20097 25631  5 328826242 13229 9315 23742  6 61 2536 2301 1442 4504  7 37 395 484 972 2149 8 25 25 25 25 25  9 25 183 744 952 1823 10 25 89 31 513 817 11 29 7082618 2165 3666 12 198 1458 1079 612 797 13 38 433 566 1080 626 14 104541 3247 1609 838 15 212 2630 2472 224 1496 16 183 2616 6680 2085 163117 28 201 375 222 1540 18 31699 15544 23095 6412 9059 19 63 243 554 299441 The table show that the highest titers were obtained for groups 4, 5and 18, in which the adjuvants were 125 μg MPL, 50 μg MPL and QS21 plusMPL.

EXAMPLE IX Therapeutic Efficacy of Different Adjuvants

A therapeutic efficacy study was conducted in PDAPP transgenic mice witha set of adjuvants suitable for use in humans to determine their abilityto potentiate immune responses to Aβ and to induce the immune-mediatedclearance of amyloid deposits in the brain.

One hundred eighty male and female, 7.5- to 8.5-month old heterozygousPDAPP transgenic mice were obtained from Charles River Laboratories. Themice were sorted into nine groups containing 15 to 23 animals per groupto be immunized with AN1792 or AN1528 combined with various adjuvants.Animals were distributed to match the gender, age, and parentage of theanimals within the groups as closely as possible. The adjuvants includedalum, MPL, and QS21, each combined with both antigens, and Freund'sadjuvant (FA) combined with only AN1792. An additional group wasimmunized with AN1792 formulated in PBS buffer plus the preservativethimerosal without adjuvant. A ninth group was immunized with PBS aloneas a negative control.

Preparation of aggregated Aβ peptides: human Aβ1-40 (AN1528; CaliforniaPeptides Inc., Napa, Calif.; Lot ME0541) and human Aβ1-42 (AN1792;California Peptides Inc., Lot ME0439) peptides were freshly solubilizedfor the preparation of each set of injections from lyophilized powdersthat had been stored desiccated at −20° C. For this purpose, two mg ofpeptide were added to 0.9 ml of deionized water and the mixture wasvortexed to generate a relatively uniform solution or suspension. AN1528was soluble at this step, in contrast to AN1792. A 100 μl aliquot of10×PBS (1×PBS: 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.5) was thenadded at which point AN1528 began to precipitate. The suspensions werevortexed again and incubated overnight at 37° C. for use the next day.

To prepare vaccine doses with alum (Groups 1 and 5). Aβ peptide in PBSwas added to Alhydrogel (two percent aqueous aluminum hydroxide gel,Sargeant, Inc., Clifton, N.J.) to reach concentrations of 100 μg Aβpeptide per 2 mg of alum. 10×PBS was added to a final dose volume of 200μl in 1×PBS. The suspension was then gently mixed for approximately 4 hrat RT prior to injection.

To prepare vaccine doses for with MPL (Groups 2 and 6), lyophilizedpowder (Ribi ImmunoChem Research, Inc., Hamilton, Mont.; Lot67039-E0896B) was added to 0.2% aqueous triethylamine to a finalconcentration of 1 mg/ml and vortexed. The mixture was heated to 65 to70° C. for 30 sec to create a slightly opaque uniform suspension ofmicelles. The solution was stored at 4° C. For each set of injections,100 μg of peptide per dose in 50 μl PBS, 50 μg of MPL per dose (50 μl)and 100 μl of PBS per dose were mixed in a borosilicate tube immediatelybefore use.

To prepare vaccine doses with QS21 (Groups 3 and 7), lyophilized powder(Aquila, Framingham, Mass.; Lot A7018R) was added to PBS, pH 6.6-6.7 toa final concentration of 1 mg/ml and vortexed. The solution was storedat −20° C. For each set of injections, 100 μg of peptide per dose in 50μl PBS, 25 μg of QS21 per dose in 25 μl PBS and 125 μl of PBS per dosewere mixed in a borosilicate tube immediately before use.

To prepare vaccine doses with Freund's Adjuvant (Group 4), 100 μg ofAN1792 in 200 μl PBS was emulsified 1:1 (vol:vol) with Complete Freund'sAdjuvant (CFA) in a final volume of 400 μl for the first immunization.For subsequent immunizations, the antigen was similarly emulsified withIncomplete Freund's Adjuvant (IFA). For the formulations containing theadjuvants alum, MPL or QS21, 100 μg per dose of AN1792 or AN1528 wascombined with alum (2 mg per dose) or MPL (50 μg per dose) or QS21 (25μg per dose) in a final volume of 200 μl PBS and delivered bysubcutaneous inoculation on the back between the shoulder blades. Forthe group receiving FA, 100 g of AN1792 was emulsified 1:1 (vol:vol)with Complete Freund's adjuvant (CFA) in a final volume of 400 μl anddelivered intraperitoneally for the first immunization, followed by aboost of the same amount of immunogen in Incomplete Freund's adjuvant(IFA) for the subsequent five doses. For the group receiving AN1792without adjuvant, 10 μg AN1792 was combined with 5 μg thimerosal in afinal volume of 50 μl PBS and delivered subcutaneously. The ninth,control group received only 200 μl PBS delivered subcutaneously.Immunizations were given on a biweekly schedule for the first threedoses, then on a monthly schedule thereafter on days 0, 16, 28, 56, 85and 112. Animals were bled six to seven days following each immunizationstarting after the second dose for the measurement of antibody titers.Animals were euthanized approximately one week after the final dose.Outcomes were measured by ELISA assay of Aβ and APP levels in brain andby immunohistochemical evaluation of the presence of amyloid plaques inbrain sections. In addition, Aβ-specific antibody titers, andAβ-dependent proliferative and cytokine responses were determined.

Table 9 shows that the highest antibody titers to Aβ1-42 were elicitedwith FA and AN1792, titers which peaked following the fourthimmunization (peak GMT: 75,386) and then declined by 59% after thefinal, sixth immunization. The peak mean titer elicited by MPL withAN1792 was 62% lower than that generated with FA (peak GMT: 28,867) andwas also reached early in the immunization scheme, after 3 doses,followed by a decline to 28% of the peak value after the sixthimmunization. The peak mean titer generated with QS21 combined withAN1792 (GMT: 1,511) was about 5-fold lower than obtained with MPL. Inaddition, the kinetics of the response were slower, since an additionalimmunization was required to reach the peak response. Titers generatedby alum-bound AN1792 were marginally greater than those obtained withQS21 and the response kinetics were more rapid. For AN1792 delivered inPBS with thimerosal the frequency and size of titers were barely greaterthan that for PBS alone. The peak titers generated with MPL and AN1528(peak GMT 3099) were about 9-fold lower than those with AN1792.Alum-bound AN1528 was very poorly immunogenic with low titers generatedin only some of the animals. No antibody responses were observed in thecontrol animals immunized with PBS alone.

TABLE 9 Geometric Mean Antibody Titers^(a) Week of Bleed Treatment 3.35.0 9.0 13.0 17.0 Alum/ 102 1,081 2,366 1,083 572 AN1792  (12/21)^(b)(17/20) (21/21) (19/21) (18/21) MPL/ 6241 28,867 1,1242 5,665 8,204AN1792 (21/21) (21/21) (21/21) (20/20) (20/20) QS21/ 30 227 327 1,5111,188 AN1792  (1/20) (10/19) (10/19) (17/18) (14/18) CFA/ 10,076 61,27975,386 41,628 30,574 AN1792 (15/15) (15/15) (15/15) (15/15) (15/15)Alum/ 25 33 39 37 31 AN1528  (0/21)  (1/21)  (3/20)  (1/20)  (2/20) MPL/184 2,591 1,653 1,156 3,099 AN1528 (15/21) (20/21) (21/21) (20/20)(20/20) QS21/ 29 221 51 820 2,994 AN1528  (1/22) (13/22)  (4/22) (20/22)(21/22) PBS plus 25 33 39 37 47 Thimer  (0/16)  (2/16)  (4/16)  (3/16) (4/16) PBS 25 25 25 25 25  (0/16)  (0/16)  (0/15)  (0/12)  (0/16)Footnotes: ^(a)Geometric mean antibody titers measured against Aβ1-42^(b)Number of responders per group

The results of AN1792 or AN1592 treatment with various adjuvants, orthimerosal on cortical amyloid burden in 12-month old mice determined byELISA are shown in FIGS. 15A-15E. In PBS control PDAPP mice the medianlevel of total Aβ in the cortex at 12 months was 1,817 ng/g (FIG. 15A).Notably reduced levels of Aβ were observed in mice treated with AN1792plus CFA/IFA (FIG. 15C), AN1792 plus alum (FIG. 15D), AN1792 plus MPL(FIG. 15E) and QS21 plus AN1792 (FIG. 15E). The reduction reachedstatistical significance (p<0.05) only for AN1792 plus CFA/IFA (FIG.15C). However, as shown in Examples I and III, the effects ofimmunization in reducing Aβ levels become substantially greater in 15month and 18 month old mice. Thus, it is expected that at least theAN1792 plus alum, AN1792 plus MPL and AN1792 plus QS21 compositions willachieve statistical significance in treatment of older mice. Bycontrast, the AN1792 plus the preservative thimerosal (FIG. 15D) showeda median level of Aβ about the same as that in the PBS treated mice.Similar results were obtained when cortical levels of Aβ42 werecompared. The median level of A42 in PBS controls was 1624 ng/g. Notablyreduced median levels of 403, 1149, 620 and 714 were observed in themice treated with AN1792 plus CFA/IFA, AN1792 plus alum, AN1792 plus MPLand AN1792 plus QS21 respectively, with the reduction achievingstatistical significance (p=0.05) for the AN1792 CFA/IFAβ treatmentgroup. The median level in the AN1792 thimerosal treated mice was 1619ng/g Aβ.

EXAMPLE X Toxicity Analysis

Tissues were collected for histopathologic examination at thetermination of studies described in Examples 2, 3 and 7. In addition,hematology and clinical chemistry were performed on terminal bloodsamples from Examples 3 and 7. Most of the major organs were evaluated,including brain, pulmonary, lymphoid, gastrointestinal, liver, kidney,adrenal and gonads. Although sporadic lesions were observed in the studyanimals, there were no obvious differences, either in tissues affectedor lesion severity, between AN1792 treated and untreated animals. Therewere no unique histopathological lesions noted in AN-1782-immunizedanimals compared to PBS-treated or untreated animals. There were also nodifferences in the clinical chemistry profile between adjuvant groupsand the PBS treated animals in Example 7. Although there weresignificant increases in several of the hematology parameters betweenanimals treated with AN1792 and Freund's adjuvant in Example 7 relativeto PBS treated animals, these type of effects are expected from Freund'sadjuvant treatment and the accompanying peritonitis and do not indicateany adverse effects from AN1792 treatment. Although not part of thetoxicological evaluation, PDAPP mouse brain pathology was extensivelyexamined as part of the efficacy endpoints. No sign of treatment relatedadverse effect on brain morphology was noted in any of the studies.These results indicate that AN1792 treatment is well tolerated and atleast substantially free of side effects.

EXAMPLE XI Prevention and Treatment of Subjects

A single-dose phase I trial is performed to determine safety. Atherapeutic agent is administered in increasing dosages to differentpatients starting from about 0.01 the level of presumed efficacy, andincreasing by a factor of three until a level of about 10 times theeffective mouse dosage is reached.

A phase II trial is performed to determine therapeutic efficacy.Patients with early to mid Alzheimer's Disease defined using Alzheimer'sdisease and Related Disorders Association (ADRDA) criteria for probableAD are selected. Suitable patients score in the 12-26 range on theMini-Mental State Exam (MMSE). Other selection criteria are thatpatients are likely to survive the duration of the study and lackcomplicating issues such as use of concomitant medications that mayinterfere. Baseline evaluations of patient function are made usingclassic psychometric measures, such as the MMSE, and the ADAS, which isa comprehensive scale for evaluating patients with Alzheimer's Diseasestatus and function. These psychometric scales provide a measure ofprogression of the Alzheimer's condition. Suitable qualitative lifescales can also be used to monitor treatment. Disease progression canalso be monitored by MRI. Blood profiles of patients can also bemonitored including assays of immunogen-specific antibodies and T-cellsresponses.

Following baseline measures, patients begin receiving treatment. Theyare randomized and treated with either therapeutic agent or placebo in ablinded fashion. Patients are monitored at least every six months.Efficacy is determined by a significant reduction in progression of atreatment group relative to a placebo group.

A second phase II trial is performed to evaluate conversion of patientsfrom non-Alzheimer's Disease early memory loss, sometimes referred to asage-associated memory impairment (AAMI), to probable Alzheimer's diseaseas defined as by ADRDA criteria. Patients with high risk for conversionto Alzheimer's Disease are selected from a non-clinical population byscreening reference populations for early signs of memory loss or otherdifficulties associated with pre-Alzheimer's symptomatology, a familyhistory of Alzheimer's Disease, genetic risk factors, age, sex, andother features found to predict high-risk for Alzheimer's Disease.Baseline scores on suitable metrics including the MMSE and the ADAStogether with other metrics designed to evaluate a more normalpopulation are collected. These patient populations are divided intosuitable groups with placebo comparison against dosing alternatives withthe agent. These patient populations are followed at intervals of aboutsix months, and the endpoint for each patient is whether or not he orshe converts to probable Alzheimer's Disease as defined by ADRDAcriteria at the end of the observation.

EXAMPLE XII General Materials and Methods

1. Measurement of Antibody Titers

Mice were bled by making a small nick in the tail vein and collectingabout 200 μl of blood into a microfuge tube. Guinea pigs were bled byfirst shaving the back hock area and then using an 18 gauge needle tonick the metatarsal vein and collecting the blood into microfuge tubes.Blood was allowed to clot for one hr at room temperature (RT), vortexed,then centrifuged at 14,000×g for 10 min to separate the clot from theserum. Serum was then transferred to a clean microfuge tube and storedat 4° C. until titered.

Antibody titers were measured by ELISA. 96-well microtiter plates(Costar EIA plates) were coated with 100 μl of a solution containingeither 10 μg/ml either Aβ42 or SAPP or other antigens as noted in eachof the individual reports in Well Coating Buffer (0.1 M sodiumphosphate, pH 8.5, 0.1% sodium azide) and held overnight at RT. Thewells were aspirated and sera were added to the wells starting at a1/100 dilution in Specimen Diluent (0.014 M sodium phosphate, pH 7.4,0.15 M NaCl, 0.6% bovine serum albumin, 0.05% thimerosal). Seven serialdilutions of the samples were made directly in the plates in three-foldsteps to reach a final dilution of 1/218,700. The dilutions wereincubated in the coated-plate wells for one hr at RT. The plates werethen washed four times with PBS containing 0.05% Tween 20. The secondantibody, a goat anti-mouse Ig conjugated to horseradish peroxidase(obtained from Boehringer Mannheim), was added to the wells as 100 μl ofa 1/3000 dilution in Specimen Diluent and incubated for one hr at RT.Plates were again washed four times in PBS, Tween 20. To develop thechromogen, 100 μl of Slow TMB (3,3′,5,5′-tetramethyl benzidine obtainedfrom Pierce Chemicals) was added to each well and incubated for 15 minat RT. The reaction was stopped by the addition of 25 μl of 2 M H₂SO₄.The color intensity was then read on a Molecular Devices Vmax at (450nm-650 nm).

Titers were defined as the reciprocal of the dilution of serum givingone half the maximum OD. Maximal OD was generally taken from an initial1/100 dilution, except in cases with very high titers, in which case ahigher initial dilution was necessary to establish the maximal OD. Ifthe 50% point fell between two dilutions, a linear extrapolation wasmade to calculate the final titer. To calculate geometric mean antibodytiters, titers less than 100 were arbitrarily assigned a titer value of25.

2. Lymphocyte Proliferation Assay

Mice were anesthetized with isoflurane. Spleens were removed and rinsedtwice with 5 ml PBS containing 10% heat-inactivated fetal bovine serum(PBS-FBS) and then homogenized in a 50μ Centricon unit (Dako A/S,Denmark) in 1.5 ml PBS-FBS for 10 sec at 100 rpm in a Medimachine (Dako)followed by filtration through a 100μ pore size nylon mesh. Splenocyteswere washed once with 15 ml PBS-FBS, then pelleted by centrifugation at200×g for 5 min. Red blood cells were lysed by resuspending the pelletin 5 mL buffer containing 0.15 M NH₄Cl, 1 M KHCO₃, 0.1 M NaEDTA, pH 7.4for five min at RT. Leukocytes were then washed as above. Freshlyisolated spleen cells (10⁵ cells per well) were cultured in triplicatesets in 96-well U-bottomed tissue culture-treated microtiter plates(Corning, Cambridge, Mass.) in RPMI 1640 medium (JRH Biosciences,Lenexa, Kans.) supplemented with 2.05 mM L glutamine, 1%Penicillin/Streptomycin, and 10% heat-inactivated FBS, for 96 hr at 37°C. Various Aβ peptides, Aβ1-16, Aβ1-40, Aβ1-42 or Aβ40-1 reversesequence protein were also added at doses ranging from 5 μM to 0.18 μMin four steps. Cells in control wells were cultured with Concanavalin A(Con A) (Sigma, cat. #C-5275, at 1 μg/ml) without added protein. Cellswere pulsed for the final 24 hr with ³H-thymidine (1 μCi/well obtainedfrom Amersham Corp., Arlington Heights Ill.). Cells were then harvestedonto UniFilter plates and counted in a Top Count MicroplateScintillation Counter (Packard Instruments, Downers Grove, Ill.).Results are expressed as counts per minute (cpm) of radioactivityincorporated into insoluble macromolecules.

3. Brain Tissue Preparation

After euthanasia, the brains were removed and one hemisphere wasprepared for immunohistochemical analysis, while three brain regions(hippocampus, cortex and cerebellum) were dissected from the otherhemisphere and used to measure the concentration of various Aβ proteinsand APP forms using specific ELISAs (Johnson-Wood et al., supra).

Tissues destined for ELISAs were homogenized in 10 volumes of ice-coldguanidine buffer (5.0 M guanidine-HCl, 50 mM Tris-HCl, pH 8.0). Thehomogenates were mixed by gentle agitation using an Adams Nutator(Fisher) for three to four hr at RT, then stored at −20° C. prior toquantitation of Aβ and APP. Previous experiments had shown that theanalytes were stable under this storage condition, and that synthetic Aβprotein (Bachem) could be quantitatively recovered when spiked intohomogenates of control brain tissue from mouse littermates (Johnson-Woodet al., supra).

4. Measurement of Aβ Levels

The brain homogenates were diluted 1:10 with ice cold Casein Diluent(0.25% casein, PBS, 0.05% sodium azide, 20 μg/ml aprotinin, 5 mM EDTA pH8.0, 10 μg/ml leupeptin) and then centrifuged at 16,000×g for 20 min at4 C. The synthetic Aβ protein standards (1-42 amino acids) and the APPstandards were prepared to include 0.5 M guanidine and 0.1% bovine serumalbumin (BSA) in the final composition. The “total” Aβ sandwich ELISAutilizes monoclonal antibody (mAb) 266, specific for amino acids 13-28of Aβ (Seubert, et al.), as the capture antibody, and biotinylated mAb3D6, specific for amino acids 1-5 of Aβ (Johnson-Wood, et al), as thereporter antibody. The 3D6 mAb does not recognize secreted APP orfull-length APP, but detects only Aβ species with an amino-terminalaspartic acid. The cell line designated RB96 3D6.32.2.4 producing theantibody 3D6 has the ATCC accession number PTA-5130, having beendeposited on Apr. 8, 2003. This assay has a lower limit of sensitivityof ˜50 ng/ml (11 nM) and shows no cross-reactivity to the endogenousmurine Aβ protein at concentrations up to 1 ng/ml (Johnson-Wood et al.,supra).

The Aβ1-42 specific sandwich ELISA employs mAβ 21F12, specific for aminoacids 33-42 of Aβ (Johnson-Wood, et al.), as the capture antibody.Biotinylated mAβ 3D6 is also the reporter antibody in this assay whichhas a lower limit of sensitivity of about 125 ρg/ml (28 ρM, Johnson-Woodet al.). For the Aβ ELISAs, 100 μl of either mAβ 266 (at 10 μg/ml) ormAβ 21F12 at (5 μl/g/ml) was coated into the wells of 96-wellimmunoassay plates (Costar) by overnight incubation at RT. The solutionwas removed by aspiration and the wells were blocked by the addition of200 μl of 0.25% human serum albumin in PBS buffer for at least 1 hr atRT. Blocking solution was removed and the plates were stored desiccatedat 4° C. until used. The plates were rehydrated with Wash Buffer[Tris-buffered saline (0.15 M NaCl, 0.01 M Tris-HCl, pH 7.5), plus 0.05%Tween 20] prior to use. The samples and standards were added intriplicate aliquots of 100 μl per well and then incubated overnight at4° C. The plates were washed at least three times with Wash Bufferbetween each step of the assay. The biotinylated mAβ 3D6, diluted to 0.5μg/ml in Casein Assay Buffer (0.25% casein, PBS, 0.05% Tween 20, pH7.4), was added and incubated in the wells for 1 hr at RT. Anavidin-horseradish peroxidase conjugate, (Avidin-HRP obtained fromVector, Burlingame, Calif.), diluted 1:4000 in Casein Assay Buffer, wasadded to the wells for 1 hr at RT. The colorimetric substrate, SlowTMB-ELISA (Pierce), was added and allowed to react for 15 minutes at RT,after which the enzymatic reaction was stopped by the addition of 25 μl2 N H₂SO₄. The reaction product was quantified using a Molecular DevicesVmax measuring the difference in absorbance at 450 nm and 650 nm.

5. Measurement of APP Levels

Two different APP assays were utilized. The first, designated APP-α/FL,recognizes both APP-alpha (α) and full-length (FL) forms of APP. Thesecond assay is specific for APP-α. The APP-α/FL assay recognizessecreted APP including the first 12 amino acids of Aβ. Since thereporter antibody (2H3) is not specific to the α-clip-site, occurringbetween amino acids 612-613 of APP695 (Esch et al., Science 248,1122-1124 (1990)); this assay also recognizes full length APP (APP-FL).Preliminary experiments using immobilized APP antibodies to thecytoplasmic tail of APP-FL to deplete brain homogenates of APP-FLsuggest that approximately 30-40% of the APP-α/FL APP is FL (data notshown). The capture antibody for both the APP-α/FL and APP-α assays ismAβ 8E5, raised against amino acids 444 to 592 of the APP695 form (Gameset al., supra). The reporter mAβ for the APP-α/FL assay is mAβ 2H3,specific for amino acids 597-608 of APP695 (Johnson-Wood et al., supra)and the reporter antibody for the APP-α assay is a biotinylatedderivative of mAβ 16H9, raised to amino acids 605 to 611 of APP. Thelower limit of sensitivity of the APP-α/FL assay is about 11 ng/ml (150ρM) (Johnson-Wood et al.) and that of the APP-α specific assay is 22ng/ml (0.3 nM). For both APP assays, mAβ 8E5 was coated onto the wellsof 96-well EIA plates as described above for mAβ 266. Purified,recombinant secreted APP-α was used as the reference standard for theAPP-α assay and the APP-α/FL assay (Esch et al., supra). The brainhomogenate samples in 5 M guanidine were diluted 1:10 in ELISA SpecimenDiluent (0.014 M phosphate buffer, pH 7.4, 0.6% bovine serum albumin,0.05% thimerosal, 0.5 M NaCl, 0.1% NP40). They were then diluted 1:4 inSpecimen Diluent containing 0.5 M guanidine. Diluted homogenates werethen centrifuged at 16,000×g for 15 seconds at RT. The APP standards andsamples were added to the plate in duplicate aliquots and incubated for1.5 hr at T. The biotinylated reporter antibody 2H3 or 16H9 wasincubated with samples for 1 hr at RT. Streptavidin-alkaline phosphatase(Boehringer Mannheim), diluted 1:1000 in specimen diluent, was incubatedin the wells for 1 hr at RT. The fluorescent substrate4-methyl-umbellipheryl-phosphate was added for a 30-min RT incubationand the plates were read on a Cytofluor™ 2350 fluorimeter(Millipore) at365 nm excitation and 450 nm emission.

6. Immunohistochemistry

Brains were fixed for three days at 4° C. in 4% paraformaldehyde in PBSand then stored from one to seven days at 4° C. in 1% paraformaldehyde,PBS until sectioned. Forty-micron-thick coronal sections were cut on avibratome at RT and stored in cryoprotectant (30% glycerol, 30% ethyleneglycol in phosphate buffer) at −20° C. prior to immunohistochemicalprocessing. For each brain, six sections at the level of the dorsalhippocampus, each separated by consecutive 240 μm intervals, wereincubated overnight with one of the following antibodies: (1) abiotinylated anti-Aβ (mAβ, 3D6, specific for human Aβ) diluted to aconcentration of 2 μg/ml in PBS and 1% horse serum; or (2) abiotinylated mAβ specific for human APP, 8E5, diluted to a concentrationof 3 μg/ml in PBS and 1.0% horse serum; or (3) a mAβ specific for glialfibrillary acidic protein (GFAP; Sigma Chemical Co.) diluted 1:500 with0.25% Triton X-100 and 1% horse serum, in Tris-buffered saline, pH 7.4(TBS); or (4) a mAβ specific for CD11b, MAC-1 antigen, (ChemiconInternational) diluted 1:100 with 0.25% Triton X-100 and 1% rabbit serumin TBS; or (5) a mAβ specific for MHC II antigen, (Pharmingen) diluted1:100 with 0.25% Triton X-100 and 1% rabbit serum in TBS; or (6) a ratmAβ specific for CD43 Pharmingen) diluted 1:100 with 1% rabbit serum inPBS or (7) a rat mAβ specific for CD45RA (Pharmingen) diluted 1:100 with1% rabbit serum in PBS; or (8) a rat monoclonal Aβ specific for CD45RB(Pharmingen) diluted 1:100 with 1% rabbit serum in PBS; or (9) a ratmonoclonal Aβ specific for CD45 (Pharmingen) diluted 1:100 with 1%rabbit serum in PBS; or (10) a biotinylated polyclonal hamster Aβspecific for CD3e (Pharmingen) diluted 1:100 with 1% rabbit serum in PBSor (11) a rat mAβ specific for CD3 (Serotec) diluted 1:200 with 1%rabbit serum in PBS; or with (12) a solution of PBS lacking a primaryantibody containing 1% normal horse serum.

Sections reacted with antibody solutions listed in 1,2 and 6-12 abovewere pretreated with 1.0% Triton X-100, 0.4% hydrogen peroxide in PBSfor 20 min at RT to block endogenous peroxidase. They were nextincubated overnight at 4° C. with primary antibody. Sections reactedwith 3D6 or 8E5 or CD3e mAβs were then reacted for one hr at RT with ahorseradish peroxidase-avidin-biotin-complex with kit components “A” and“B” diluted 1:75 in PBS (Vector Elite Standard Kit, Vector Labs,Burlingame, Calif.). Sections reacted with antibodies specific forCD45RA, CD45RB, CD45, CD3 and the PBS solution devoid of primaryantibody were incubated for 1 hour at RT with biotinylated anti-rat IgG(Vector) diluted 1:75 in PBS or biotinylated anti-mouse IgG (Vector)diluted 1:75 in PBS, respectively. Sections were then reacted for one hrat RT with a horseradish peroxidase-avidin-biotin-complex with kitcomponents “A” and “B” diluted 1:75 in PBS (Vector Elite Standard Kit,Vector Labs, Burlingame, Calif.).

Sections were developed in 0.01% hydrogen peroxide, 0.05%3,3′-diaminobenzidine (DAB) at RT. Sections destined for incubation withthe GFAP-, MAC-1- AND MHC II-specific antibodies were pretreated with0.6% hydrogen peroxide at RT to block endogenous peroxidase thenincubated overnight with the primary antibody at 4° C. Sections reactedwith the GFAP antibody were incubated for 1 hr at RT with biotinylatedanti-mouse IgG made in horse (Vector Laboratories; Vectastain Elite ABCKit) diluted 1:200 with TBS. The sections were next reacted for one hrwith an avidin-biotin-peroxidase complex (Vector Laboratories;Vectastain Elite ABC Kit) diluted 1:1000 with TBS. Sections incubatedwith the MAC-1- or MHC II-specific mAβ as the primary antibody weresubsequently reacted for 1 hr at RT with biotinylated anti-rat IgG madein rabbit diluted 1:200 with TBS, followed by incubation for one hr withavidin-biotin-peroxidase complex diluted 1:1000 with TBS. Sectionsincubated with GFAP-, MAC-1- and MHC II-specific antibodies were thenvisualized by treatment at RT with 0.05% DAB, 0.01% hydrogen peroxide,0.04% nickel chloride, TBS for 4 and 11 min, respectively;

Immunolabeled sections were mounted on glass slides (VWR, Superfrostslides), air dried overnight, dipped in Propar (Anatech) and overlaidwith coverslips using Permount (Fisher) as the mounting medium.

To counterstain Aβ plaques, a subset of the GFAP-positive sections weremounted on Superfrost slides and incubated in aqueous 1% Thioflavin S(Sigma) for 7 min following immunohistochemical processing. Sectionswere then dehydrated and cleared in Propar, then overlaid withcoverslips mounted with Permount.

7. Image Analysis

A Videometric 150 Image Analysis System (Oncor, Inc., Gaithersburg, Md.)linked to a Nikon Microphot-FX microscope through a CCD video camera anda Sony Trinitron monitor was used for quantification of theimmunoreactive slides. The image of the section was stored in a videobuffer and a color-and saturation-based threshold was determined toselect and calculate the total pixel area occupied by the immunolabeledstructures. For each section, the hippocampus was manually outlined andthe total pixel area occupied by the hippocampus was calculated. Thepercent amyloid burden was measured as: (the fraction of the hippocampalarea containing Aβ deposits immunoreactive with mAβ 3D6)×100. Similarly,the percent neuritic burden was measured as: (the fraction of thehippocampal area containing dystrophic neurites reactive with mAβ8E5)×100. The C-Imaging System (Compix, Inc., Cranberry Township, Pa.)operating the Simple 32 Software Application program was linked to aNikon Microphot-FX microscope through an Optronics camera and used toquantitate the percentage of the retrospenial cortex occupied byGFAP-positive astrocytes and MAC-1- and MHC II-positive microglia. Theimage of the immunoreacted section was stored in a video buffer and amonochrome-based threshold was determined to select and calculate thetotal pixel area occupied by immunolabeled cells. For each section, theretrosplenial cortex (RSC) was manually outlined and the total pixelarea occupied by the RSC was calculated. The percent astrocytosis wasdefined as: (the fraction of RSC occupied by GFAP-reactiveastrocytes)×100. Similarly, percent microgliosis was defined as: (thefraction of the RSC occupied by MAC-1- or MHC II-reactivemicroglia)×100. For all image analysis, six sections at the level of thedorsal hippocampus, each separated by consecutive 240 μm intervals, werequantitated for each animal. In all cases, the treatment status of theanimals was unknown to the observer.

Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications may be practiced within the scope of the appended claims.All publications and patent documents cited herein are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted.

1. A method of therapeutically treating a disease characterized by anamyloid deposit of Aβ in a patient, comprising: administering a dosageof an Aβ peptide greater than 10 μg and an adjuvant in a regimeeffective to induce an immune response comprising antibodies to the Aβpeptide, the adjuvant enhancing the immune response to the Aβ peptide,and thereby therapeutically treat the disease in the patient.
 2. Themethod of claim 1, wherein the dose of the Aβ peptide administered tothe patient is at least 20 μg.
 3. The method of claim 1, wherein thedose of the Aβ peptide administered to the patient is at least 50 μg. 4.The method of claim 1, wherein the dose of the Aβ peptide administeredto the patient is at least 100 μg.
 5. The method of claim 1, wherein thepatient is a human.
 6. The method of claim 1, wherein the disease isAlzheimer's disease.
 7. The method of any one of claims 1-6, wherein thepatient is asymptomatic.
 8. The method of any one of claims 1-6, whereinthe patient is under
 50. 9. The method of any one of claims 1-6, whereinthe patient has inherited risk factors indicating susceptibility toAlzheimer's disease.
 10. The method of any one of claims 1-6, whereinthe patient has no known risk factors for Alzheimer's disease.
 11. Themethod of any one of claims 1-6, wherein the Aβ peptide is administeredin aggregated form.
 12. The method of any one of claims 1-6, wherein theAβ peptide is administered orally, subcutaneously, intramuscularly,topically or intravenously.
 13. The method of any one of claims 1-6,wherein the Aβ peptide is administered intramuscularly orsubcutaneously.
 14. The method of claim 1, wherein the adjuvant and theAβ peptide are administered together as a composition.
 15. The method ofclaim 1, wherein the adjuvant is administered before the Aβ peptide. 16.The method of claim 1, wherein the adjuvant is administered after the Aβpeptide.
 17. The method of claim 1, wherein the adjuvant is alum. 18.The method of claim 1, wherein the adjuvant is MPL.
 19. The method ofclaim 1, wherein the adjuvant is QS21.
 20. The method of claim 1,wherein the adjuvant is M-CSF.
 21. The method of any one of claims 1-6,wherein the Aβ peptide is administered wit GM-CSF in the regime.
 22. Amethod of prophylaxis of a disease characterized by an amyloid depositof Aβ in a patient, comprising: administering a dosage of an Aβ peptidegreater than 10 μg and an adjuvant in a regime effective to induce animmune response comprising antibodies to the Aβ peptide, the adjuvantenhancing the immune response to the Aβ peptide, and thereby effectprophylaxis of the disease in the patient.
 23. The method of claim 22,wherein the dose of the Aβ peptide administered to the patient is atleast 20 μg.
 24. The method of claim 22, wherein the dose of the Aβpeptide administered to the patient is at least 50 μg.
 25. The method ofclaim 22, wherein the dose of the Aβ peptide administered to the patientis at least 100 μg.
 26. The method of claim 22, wherein the patient is ahuman.
 27. The method of claim 22, wherein the disease is Alzheimer'sdisease.
 28. The method of any one of claims 22-27, wherein the patientis asymptomatic.
 29. The method of any one of claims 22-27, wherein thepatient is under
 50. 30. The method of any one of claims 22-27, whereinthe patient has inherited risk factors indicating susceptibility toAlzheimer's disease.
 31. The method of any one of claims 22-27, whereinthe patient has no known risk factors for Alzheimer's disease.
 32. Themethod of any one of claims 22-27, wherein the Aβ peptide isadministered in aggregated form.
 33. The method of any one of claims22-27, wherein the Aβ peptide is administered orally, subcutaneously,intramuscularly, topically or intravenously.
 34. The method of any oneof claims 22-27, wherein the Aβ peptide is administered intramuscularlyor subcutaneously.
 35. The method of claim 22, wherein the adjuvant andthe Aβ peptide are administered together as a composition.
 36. Themethod of claim 22, wherein the adjuvant is administered before the Aβpeptide.
 37. The method of claim 22, wherein the adjuvant isadministered after the Aβ peptide.
 38. The method of claim 22, whereinthe adjuvant is alum.
 39. The method of claim 22, wherein the adjuvantis MPL.
 40. The method of claim 22, wherein the adjuvant is QS21. 41.The method of claim 22, wherein the adjuvant is M-CSF.
 42. The method ofany one of claims 22-27, wherein the Aβ peptide is administered withGM-CSF in the regime.
 43. A method of therapeutically treating a diseasecharacterized by an amyloid deposit of Aβ in a patient, comprising:administering an Aβ peptide in a regime effective to induce an immuneresponse comprising antibodies to the Aβ peptide and therebytherapeutically treat the disease in the patient, wherein the dose ofthe Aβ peptide administered to the patient is at least 50 μg.
 44. Themethod of claim 43, wherein the patient is a human.
 45. The method ofclaim 43, wherein the disease is Alzheimer's disease.
 46. The method ofany one of claims 43-45, wherein the patient is asymptomatic.
 47. Themethod of any one of claims 43-45, wherein the patient is under
 50. 48.The method of any one of claims 43-45, wherein the patient has inheritedrisk factors indicating susceptibility to Alzheimer's disease.
 49. Themethod of any one of claims 43-45, wherein the patient has no known riskfactors for Alzheimer's disease.
 50. The method of any one of claims43-45, wherein the Aβ peptide is administered in aggregated form. 51.The method of any one of claims 43-45, wherein the Aβ peptide isadministered orally, subcutaneously, intramuscularly, topically orintravenously.
 52. The method of any one of claims 43-45, wherein the Aβpeptide is administered intramuscularly or subcutaneously.
 53. Themethod of any one of claims 43-45, further comprising administering anadjuvant, wherein the adjuvant enhances the immune response to the Aβpeptide.
 54. The method of claim 53, wherein the adjuvant and the Aβpeptide are administered together as a composition.
 55. The method ofclaim 54, wherein the adjuvant is administered before the Aβ peptide.56. The method of claim 54, wherein the adjuvant is administered afterthe Aβ peptide.
 57. The method of claim 54, wherein the adjuvant isalum.
 58. The method of claim 54, wherein the adjuvant is MPL.
 59. Themethod of claim 54, wherein the adjuvant is QS21.
 60. The method ofclaim 54, wherein the adjuvant is M-CSF.
 61. The method of any one ofclaims 43-45, wherein the Aβ peptide is administered with GM-CSF in theregime.
 62. A method of prophylaxis of a disease characterized by anamyloid deposit of Aβ in a patient, comprising: administering an Aβpeptide in a regime effective to induce an immune response comprisingantibodies to the Aβ peptide and thereby effect prophylaxis of thedisease in the patient, wherein the dose of the Aβ peptide administeredto the patient is at least 50 μg.
 63. The method of claim 62, whereinthe patient is a human.
 64. The method of claim 62, wherein the diseaseis Alzheimer's disease.
 65. The method of any one of claims 62-64,wherein the patient is asymptomatic.
 66. The method of any one of claims62-64, wherein the patient is under
 50. 67. The method of any one ofclaims 62-64, wherein the patient has inherited risk factors indicatingsusceptibility to Alzheimer's disease.
 68. The method of any one ofclaims 62-64, wherein the patient has no known risk factors forAlzheimer's disease.
 69. The method of any one of claims 62-64, whereinthe Aβ peptide is administered in aggregated form.
 70. The method of anyone of claims 62-64, wherein the Aβ peptide is administered orally,subcutaneously, intramuscularly, topically or intravenously.
 71. Themethod of any one of claims 62-64, wherein the Aβ peptide isadministered intramuscularly or subcutaneously.
 72. The method of anyone of claims 62-64, further comprising administering an adjuvant,wherein the adjuvant enhances the immune response to the Aβ peptide. 73.The method of claim 72, wherein the adjuvant and the Aβ peptide areadministered together as a composition.
 74. The method of claim 72,wherein the adjuvant is administered before the Aβ peptide.
 75. Themethod of claim 72, wherein the adjuvant is administered after the Aβpeptide.
 76. The method of claim 72, wherein the adjuvant is alum. 77.The method of claim 72, wherein the adjuvant is MPL.
 78. The method ofclaim 72, wherein the adjuvant is QS21.
 79. The method of claim 72,wherein the adjuvant is M-CSF.
 80. The method of any one of claims62-64, wherein the Aβ peptide is administered with GM-CSF in the regime.